Crown lengthening procedure in the management of amelogenesis imperfecta.

Full mouth rehabilitation includes a promising treatment planning and execution thus fulfilling esthetic, occlusal, and functional parameters maintaining the harmony of the stomatognathic system. Crown lengthening procedures have become an integral component of the esthetic armamentarium and are utilized with increasing frequency to enhance the appearance of restorations placed in the esthetic zone. Crown lengthening plays a role to create healthy relationship of the gingiva and bone levels so as to gain access to more of the tooth which can be restored, if it is badly worn, decayed or fractured, below the gum line. This paper highlights the full mouth crown lengthening procedure performed on a patient with amelogenesis imperfecta.

Evidence for a potential tumor suppressor role for the Na,K-ATPase beta1-subunit.

The Na,K-ATPase, consisting of two essential subunits (alpha, beta), plays a critical role in the regulation of ion homeostasis in mammalian cells. Recent studies indicate that reduced expression of the beta1 isoform (NaK-beta1) is commonly observed in carcinoma and is associated with events involved in cancer progression. In this study, we present evidence that repletion of NaK-beta1 in Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK), a highly tumorigenic cell line, inhibits anchorage independent growth and suppresses tumor formation in immunocompromised mice. Additionally, using an in vitro cell-cell aggregation assay, we showed that cell aggregates of NaK-beta1 subunit expressing MSV-MDCK cells have reduced extracellular regulated kinase (ERK) 1/2 activity compared with parental MSV-MDCK cells. Finally, using immunohistochemistry and fully quantitative image analysis approaches, we showed that the levels of phosphorylated ERK 1/2 are inversely correlated to the NaK-beta1 levels in the tumors. These findings reveal for the first time that NaK-beta1 has a potential tumor-suppressor function in epithelial cells.

Identification of protein kinase C as an intermediate in Na,K-ATPase beta-subunit mediated lamellipodia formation and suppression of cell motility in carcinoma cells.

We have shown that repletion of Na,K-ATPase Beta1-subunit (Na,K-Beta) in Moloney Sarcoma virus transformed MDCK (MSV-Na,K-Beta) cells induced lamellipodia and suppressed motility in a PI3-Kinase dependent manner. In this study, we provide evidence that decreased cell motility is due to increased attachment of Na,K-Beta expressing cells to the substratum. Treatment of MSV-Beta-GFP cells with bisindolylmalemide, a general Protein Kinase C (PKC) inhibitor, abolished PI3-Kinase activation and its down stream effects of Rac1 activation, binding of Na,K-Beta to annexin II, and suppression of cell motility and attachment. Thus, these studies unraveled that a PKC is involved upstream of PI3-Kinase in the suppression of Na,K-Beta mediated cell motility in carcinoma cells.

Renal clear-cell carcinoma: an ultrastructural study on the junctional complexes.

Junctional complexes such as tight junctions, adherens junctions, and desmosomes play crucial roles in the structure and function of epithelial cells. These junctions are involved in increasing cell-cell contact and as well serve as signaling centers regulating multiple functions in epithelial cells. Carcinoma cell lines cultured in the laboratory generally lack junctional complexes. However, studies directed towards understanding the distribution of junctional complexes in human cancer tissues are lacking. In this study, we analyzed by electron microscopy the distribution of junctional complexes in patients diagnosed with renal clear-cell carcinoma. We found that both tight junctions and adherens junctions were drastically reduced in patients with cancer compared to normal tissues. Desmosomes were not detected in normal proximal tubules while distinctly present in cancer tissues. These results suggest that analysis of junctional complexes in human tumors should provide valuable information that might have prognostic and diagnostic value.

Na,K-ATPase activity is required for formation of tight junctions, desmosomes, and induction of polarity in epithelial cells.

Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na(+) homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.

Na,K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility.

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an alpha- and beta-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin-mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and beta(1)-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase beta(1)-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin-mediated cell-cell adhesion requires the Na,K-ATPase beta-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the beta(1)-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.

Reduced expression of beta-subunit of Na,K-ATPase in human clear-cell renal cell carcinoma.

PURPOSE: Multiple subtypes of renal cancer have been identified. Clear-cell renal cell carcinoma (RCC) is the most common subtype of RCC and one of the more aggressive. The goal of this study was to investigate in RCC the levels of Na,K-ATPase, an abundant enzyme in the kidney which is crucial for various kidney functions. Na,K-ATPase is a heterodimer consisting of a catalytic a-subunit and a glycosylated beta-subunit whose function is still not well-defined. MATERIALS AND METHODS: Fourteen clear-cell RCC specimens were studied. The levels of the Na,K-ATPase alpha and beta-subunits in normal kidney and RCC tissues were determined by immunoblot analysis. The localization of the alpha and beta-subunits was studied by immunofluorescence and laser scanning confocal microscopy. Na,K-ATPase activity was determined using a coupled-enzyme spectrophotometric assay. RESULTS: In normal kidney, the cells demonstrate an epithelial morphology with distinct basolateral plasma membrane localization of the alpha and beta-subunits. Conversely, the cells of the clear-cell RCC have lost their epithelial phenotype and the alpha and beta-subunits show a diffuse intracellular staining. Clear-cell RCC tumor cell lysates showed a consistent 95.6+/-2.8% (mean +/- SD) reduction in protein levels of beta-subunit relative to the levels in normal kidney. The alpha-subunit level in RCC lysates was generally near or above the levels relative to normal kidney. The reduced beta-subunit expression was accompanied by a significant reduction in the Na,K-ATPase activity in RCC membranes. CONCLUSIONS: These results suggest that the beta-subunit may regulate the Na,K-ATPase activity in vivo. Diminished Na,K-ATPase activity in conjunction with the reduced beta-subunit level is associated with the clear-cell RCC phenotype.

Only the first and the last hydrophobic segments in the COOH-terminal third of Na,K-ATPase alpha subunit initiate and halt, respectively, membrane translocation of the newly synthesized polypeptide. Implications for the membrane topology.

We studied the topogenic properties of five hydrophobic segments (H5-H9) in the COOH-terminal third of Na,K-ATPase alpha subunit using in vitro insertion of fusion proteins into endoplasmic reticulum membranes. These fusion proteins consisted of several different lengths of truncated alpha subunit starting at Met729 and a reporter protein, chloramphenicol acetyltransferase, that was linked in frame after each hydrophobic segment. We found that membrane insertion of the newly synthesized COOH-terminal third was initiated by H5 and terminated by H9, indicating that here only H5 and H9 have topogenic function. The other three, H6-H8, did not have topogenic function in the native context and were translocated into the endoplasmic reticulum lumen. These results were in striking contrast to the previous models in which four or six hydrophobic segments were proposed to cross the membrane. Furthermore, the findings suggest a novel mechanism for achieving the final membrane topology of the COOH-terminal third of the alpha subunit.

Activation of Tsk and Btk tyrosine kinases by G protein beta gamma subunits.

Tsk/Itk and Btk are members of the pleckstrin-homology (PH) domain-containing tyrosine kinase family. The PH domain has been demonstrated to be able to interact with beta gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) (G beta gamma) and phospholipids. Using cotransfection assays, we show here that the kinase activities of Tsk and Btk are stimulated by certain G beta gamma subunits. Furthermore, using an in vitro reconstitution assay with purified bovine brain G beta gamma subunits and the immunoprecipitated Tsk, we find that Tsk kinase activity is increased by G beta gamma subunits and another membrane factor(s). These results indicate that this family of tyrosine kinases could be an effector of heterotrimeric G proteins.

A simple biochemical approach to quantitate rough endoplasmic reticulum.

In this report we demonstrate that the changes in size of the rough endoplasmic reticulum (RER) can be determined by quantifying the membrane-bound ribosomal population separated by cell fractionation and sucrose density gradient analysis. Total cell membranes, rather than microsomes, were used as the source of membrane-bound ribosomes to eliminate potential losses during the preparation of microsomes. Bound ribosomes were assayed after quantitative release and recovery from total cell membranes using puromycin in the presence of high-salt buffer. Using this analysis, we demonstrate a 4.2-fold increase in RER in estrogen-treated male Xenopus laevis liver. Furthermore, we show that the ratio of the distribution of free to membrane-bound ribosomes in a nonsecretory cell line (HeLa) was 3.3, while this ratio in a secretory cell line (AR42J) was 1.2, indicating that cells active in secretion contain more RER. We suggest that this biochemical technique provides a simpler assay to detect changes in the size of the RER.