Non-calcifying variant of calcifying epithelial odontogenic tumor with Langerhans cells.

Calcifying epithelial odontogenic tumor (CEOT) is a rare type of odontogenic tumor. The most characteristic feature of the classical CEOT is the presence of amyloid globules and Liesegang ring calcification in the tumor tissue. Here, we present a non-calcifying variant of intraosseous CEOT with the presence of Langerhans cells within tumor epithelial nests in a 52-year-old Taiwanese woman. The patient was referred from a local dentist to our hospital for treatment of a unilocular radiolucent lesion at the right anterior region of the maxilla. The lesion was excised. Microscopically, the tumor was composed of small nests or strands of odontogenic epithelial cells and amorphous eosinophilic globules of amyloid-like materials in a loose fibrous connective tissue stroma. The tumor epithelial cells were positive for pan-cytokeratins (AE1 and AE3). Langerhans cells demonstrated by anti-CD1a staining were found in nests or strands of tumor epithelial cells. The eosinophilic globules were positive for Congo red and showed green birefringence when subjected to polarized light. Review of the English literature revealed two cases of non-calcifying variant of intraosseous CEOT with Langerhans cells in the anterior and premolar regions of the maxilla. Taken together, we suggest that the non-calcifying, Langerhan cell-rich variant of CEOT may have a distinct predilection for occurrence in the anterior and premolar region of the maxilla in contrast to the classical CEOTs that usually occur in the molar and ascending ramus area of the mandible.

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Tumor necrosis factor-alpha mediates both apoptotic cell death and cell proliferation in a human hematopoietic cell line dependent on mitotic activity and receptor subtype expression.

The TF-1 human erythroleukemic cell line exhibits opposing physiological responses toward tumor necrosis factor-alpha (TNF) treatment, dependent upon the mitotic state of the cells. Mitotically active cells in log growth respond to TNF by rapidly undergoing apoptosis whereas TNF exposure stimulates cellular proliferation in mitotically quiescent cells. The concentration-dependent TNF-induced apoptosis was monitored by cellular metabolic activity and confirmed by both DNA epifluorescence and DNA fragmentation. Moreover, these responses could be detected by measuring extracellular acidification activity, enabling rapid prediction (within approximately 1.5 h of TNF treatment) of the fate of the cell in response to TNF. Growth factor resupplementation of quiescent cells, resulting in reactivation of cell cycling, altered TNF action from a proliferative stimulus to an apoptotic signal. Expression levels of the type II TNF receptor subtype (p75TNFR) were found to correlate with sensitivity to TNF-induced apoptosis. Pretreatment of log growth TF-1 cells with a neutralizing anti-p75TNFR monoclonal antibody inhibited TNF-induced apoptosis by greater than 80%. Studies utilizing TNF receptor subtype-specific TNF mutants and neutralizing antisera implicated p75TNFR in TNF-dependent apoptotic signaling. These data show a bifunctional physiological role for TNF in TF-1 cells that is dependent on mitotic activity and controlled by the p75TNFR.

Signal transduction mediated by the truncated trkB receptor isoforms, trkB.T1 and trkB.T2.

The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced "truncated receptors" with identical extracellular ligand-binding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1- and trkB. T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

Direct measurement of extracellular proton flux from isolated gastric glands.

We used the microphysiometer, a sensitive extracellular pH sensor, to resolve luminal (or apical) H+ secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Stimulation of glands via the adenosine 3',5'-cyclic monophosphate pathway produced a biphasic change in the extracellular acidification rate (EAR): after an initial transient decrease below the unstimulated baseline (-40.9 +/- 3.4%), the EAR increased to a steady-state maximal plateau (+98.1 +/- 5.3%) within 30 min (n = 37). We interpret the biphasic EAR profile as an initial excess of basolaterally released OH- followed by delayed luminal efflux of simultaneously produced H+. The elevated EAR at steady state reflected liberation of metabolic acid attributed to H(+)-K(+)-ATPase enzymatic activity. The presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid prevented OH- release and reduced steady-state EAR. Basolateral OH- release and steady-state EAR were also inhibited by the H(+)-K(+)-ATPase inactivators omeprazole and SCH-28080. Inhibition of Na+/H+ exchange did not reduce steady-state EAR and did not affect apical H+ production, as judged by the accumulation of the weak base aminopyrine. Sodium thiocyanate (1 mM), which short circuits intraluminal H+ accumulation, blocked OH- release, demonstrating its dependence on H(+)-OH- separation at the apical membrane. A computerized model was developed to illustrate how the observed biphasic EAR profile would result from a delayed luminal efflux of H+ due to transitory intraluminal compartmentalization.

A metabolic view of receptor activation in cultured cells following cryopreservation.

The effect of cryopreservation on agonist-induced receptor activation in mammalian cells was investigated with the Cytosensor microphysiometer, a biosensor that monitors cellular metabolic activity by measuring changes in extracellular pH. In this study, two different cell types--nonadherent TF-1 cells (from a human erythroleukemia patient) and adherent WT3 cells (CHO-K1 cells transfected with the m1 muscarinic acetylcholine receptor)--were cryopreserved by freezing in a disposable cell capsule used in the microphysiometer. The recovery of metabolic activity by TF-1 cells was observed over approximately 1 h following thawing. Responses of the TF-1 cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet activating factor (PAF) were measured before cryopreservation and 90 min after thawing. The GM-CSF and PAF responses retained 71 +/- 14% and 73 +/- 10% of maximum stimulation, respectively. Post-thaw cholinergic stimulation of WT3 cells was 73 +/- 9% of its level in similarly treated but unfrozen cells. Cryopreservation caused no detectable difference in desensitization of the response due to repeated application of carbachol. These results demonstrate the feasibility of pharmacological studies with cryopreserved cells in the microphysiometer and further suggest that the microphysiometer may be useful in exploring the biological consequences of cryopreservation in the early post-thaw period.

PKC epsilon is involved in granulocyte-macrophage colony-stimulating factor signal transduction: evidence from microphysiometry and antisense oligonucleotide experiments.

We have used microphysiometry and antisense methodology to show that the epsilon isoenzyme of protein kinase C (PKC) is involved in the signal transduction pathway of granulocyte-macrophage colony-stimulating factor (GM-CSF) in a human bone marrow cell line, TF-1. These cells require GM-CSF or a related cytokine for proliferation. When the cells are appropriately exposed to GM-CSF, they exhibit a burst of metabolic activity that can be detected on the time scale of minutes in the microphysiometer, a biosensor-based instrument that measures the rate at which cells excrete protons. These cells express PKC alpha and -epsilon, as determined by Western blot analysis. Treatment with isoenzyme-specific antisense oligonucleotides inhibits expression appropriately, but only inhibition of PKC epsilon appreciably diminishes the burst of metabolic activity induced by GM-CSF. Consistent with the involvement of PKC epsilon, GM-CSF appears to activate phospholipase D and does not cause a detectable increase in cytosolic [Ca2+].