Ultrasound for the detection of the pyramidal lobe of the thyroid gland.

PURPOSE: The pyramidal lobe (PL) is an ancillary lobe of the thyroid gland that can be affected by the same pathologies as the rest of the gland. We aimed to assess the diagnostic performance of high-resolution sonography in the detection of the PL with verification by dissection and histological examination. METHODS: In a prospective, cross-sectional mono-center study, 50 fresh, non-embalmed cadavers were included. Blinded ultrasound examination was performed to detect the PL by two investigators of different experience levels. If the PL was detected with ultrasound, dissection was performed to expose the PL and obtain a tissue sample. When no PL was detected with ultrasound, a tissue block of the anterior cervical region was excised. An endocrine pathologist microscopically examined all tissue samples and tissue blocks for the presence of thyroid parenchyma. RESULTS: The prevalence of the PL was 80% [40/50; 95% CI (68.9%; 91.1%)]. Diagnostic performance for both examiners was: sensitivity (85.0%; 42.5%), specificity (50.0%; 60.0%), positive predictive value (87.2%; 81.0%), negative predictive value (45.5%; 21.0%) and accuracy (78.0%; 46.0%). Regression analysis demonstrated that neither thyroid parenchyma echogenicity, thyroid gland volume, age nor body size proved to be covariates in the accurate detection of a PL (p > .05). CONCLUSION: We report that high-resolution ultrasound is an adequate examination modality to detect the PL. Our findings indicate a higher prevalence than previously reported. Therefore, the PL may be regarded as a regular part of the thyroid gland. We also advocate a dedicated assessment of the PL in routine thyroid ultrasound.

Ras mediates cell survival by regulating tuberin.

Mutational activation of Ras promotes oncogenesis by controlling cell cycle regulation and cell survival. Ras-mediated activation of both, the PI3K/AKT pathway and the MEK/ERK pathway, can trigger downregulation of the function of tuberin to block the activities of mTOR and p70S6K. Here we demonstrate that Ras-induced cell survival is accompanied by upregulation of p70S6K activity. Ras harbors the potential to negatively affect tuberin-induced apoptosis and p70S6K inactivation. These effects of Ras were found to depend on its potential to regulate the MEK/ERK pathway. Experiments using tuberin-negative fibroblasts revealed that the potential of Ras to counteract apoptosis depends on functional tuberin. Taken together, we provide evidence that the function of Ras to trigger inactivation of tuberin plays a major role in the regulation of cell survival upon mutational activation of the oncogene Ras. This is the first description of a functional interaction between the tumor suppressor tuberin and the oncogene Ras in regulating apoptosis.

Human amniotic fluid stem cells: a new perspective.

The discovery of amniotic fluid stem cells initiated a new and very promising field in stem cell research. In the last four years amniotic fluid stem cells have been shown to express markers specific to pluripotent stem cells, such as Oct-4. Due to their high proliferation potential, amniotic fluid stem cell lineages can be established. Meanwhile, they have been shown to harbor the potential to differentiate into cells of all three embryonic germ layers. It will be a major aim for the future to define the potential of this new source of stem cells for therapies related to specific diseases.

Nuclear/cytoplasmic localization of Akt activity in the cell cycle.

The serine/threonine protein kinase Akt (also known as PKB) is a proto-oncogene and one of the most frequently hyperactivated kinases in human cancer. Its activation downstream of growth-factor-stimulated phosphatidylinositide-3'-OH kinase activity plays a role in the control of cell cycle, cell growth, apoptosis and cell energy metabolism. Akt phosphorylates some thousand downstream substrates, including typical cytoplasmic as well as nuclear proteins. Accordingly, it is not surprising that Akt activity can be found in both, the cytoplasm and the nucleus. Here we report the cell cycle regulation of nuclear and cytoplasmic Akt activity in mammalian cells. These data provide new insights into the regulation of Akt activity and have implications for future studies on the regulation of the wide variety of different nuclear and cytoplasmic Akt substrates.

Akt regulates nuclear/cytoplasmic localization of tuberin.

The autosomal dominantly inherited disease tuberous sclerosis (TSC) affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. TSC1, encoding hamartin, and TSC2, encoding tuberin are tumor suppressor genes responsible for TSC. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR)/p70S6K cascade. Phosphorylation of S939 and T1462 by Akt downregulates tuberin's potential to inhibit mTOR/p70S6K. Here, we show that this tuberin phosphorylation by Akt does not affect tuberin-mediated control of p27 protein amounts. This demonstrates that regulating p27 protein amounts and mTOR/p70S6K are separable functions of tuberin. Furthermore, we found that phosphorylation by Akt triggers upregulation of cytoplasmic and downregulation of nuclear tuberin. In cycling cells with high Akt activity, tuberin is predominantly localized to the cytoplasm. In arrested G0 cells with downregulated Akt activity, a significant proportion of tuberin is localized to the nucleus. Upon re-entry into the normal ongoing cell cycle, nuclear localization of tuberin is downregulated parallel to the activation of Akt. Recently, the mTOR/p70S6K cascade has been demonstrated to exist in both the cytoplasm and nucleus. We here also found that tuberin harbors the potential to regulate p70S6K activity in both the cytoplasm and nucleus. This description of functional tuberin in the cytoplasm and the nucleus together with our observation of Akt-controlled and cell cycle-regulated tuberin localization are of particular interest for a further understanding of tuberin's function as a gate keeper of the G0 cell status as well as of Akt's activity to control cell proliferation.

Tuberin negatively affects BCL-2's cell survival function.

Uncontrolled cell cycle progression and cell growth are key properties of tumor cells. The tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC) have been demonstrated to control both, cell cycle and cell size regulation. Hamartin, encoded by TSC1, and tuberin, encoded by TSC2, form a complex, of which tuberin is assumed to be the functional component. Loss of TSC genes function triggers hamartoma development in TSC patients. However, in vivo mostly tumor cell development is rapidly terminated via apoptosis. BCL-2, the founding member of the BCL-2 family of proteins, is well known for its anti-apoptotic properties. Here we show that pro-apoptotic actinomycin D cannot interfere with BCL-2's cell survival functions. However, we found tuberin to negatively regulate BCL-2's anti-apoptotic effects on low serum-induced apoptosis. These findings warrant further investigations to elucidate the molecular mechanism underlying tuberin's negative effects on cell survival.

Tuberin activates the proapoptotic molecule BAD.

TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.

Proteins interacting with the tuberous sclerosis gene products.

Tuberous sclerosis (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 to 10000 individuals. The genes, TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for TSC. Since their identification 1997 and 1993 respectively, a variety of different functions have been described for the TSC gene products. Hamartin and tuberin form a complex, providing a tentative explanation for the similar disease phenotype in TSC patients with mutations in either of these genes. In addition, associations of hamartin or tuberin with several different proteins have been demonstrated. In this review, we summarize the current knowledge on hamartin- and tuberin-interacting proteins and discuss their role for the understanding of the functions of the TSC gene products.

Cell cycle and cell size regulation in Down syndrome cells.

Although the neuropathological features typical for Down Syndrome obviously result from deregulation of both, cell cycle control and differentiation processes, so far research focused on the latter. Considering the known similarities between the neuropathology of Down Syndrome and Alzheimer's disease and the knowledge, that in Alzheimer's disease neuronal degeneration is associated with the activation of mitogenic signals and cell cycle activation, it is tempting to investigate the consequences of an additional chromosome 21 on mammalian cell cycle regulation. We analysed the distribution of cells in different cell cycle phases on the flowcytometer and the cell size of human amniotic fluid cells with normal karyotypes and with trisomy 21. We could not detect any significant differences suggesting that the presence of an additional copy of the about 225 genes on human chromosome 21 does not trigger cell cycle effects in amniotic fluid cells. These data provide new insights into the cell biology of trisomy 21 cells.

Stem cell marker expression in human trisomy 21 amniotic fluid cells and trophoblasts.

Down Syndrome is the most frequent genetic cause of mental retardation. Deregulation of specific differentiation processes is a major cause for the neuropathological cell features typical for this syndrome. The molecular mechanisms leading to Down Syndrome are likely to be operative from the very earliest time of embryonic/fetal development. We therefore analysed human amniotic fluid cell samples and cytotrophoblastic cells from placental biopsies, both with normal karyotypes and with trisomy 21, for the mRNA expression of stem cell marker genes. Here we describe for the first time that these human primary cell sources contain cells that express telomerase reverse transcriptase, leukemia inhibitory factor receptor, and bone morphogenetic protein receptor II. A specific difference between aneuploid and normal cells could not be detected. These data provide evidence that human amniotic fluid and cytotrophoblastic cell cultures might provide a new source for research on primary cell systems expressing these stem cell markers. In addition, it is suggested that early deregulation of the expression of these genes in the here analysed cell sources does not contribute to the molecular development of Down Syndrome.

Cloning and expression of cDNAs encoding alpha1,3-fucosyltransferase homologues from Arabidopsis thaliana.

The core alpha1,3-fucosyltransferases are involved in the synthesis of glycans specific to plants and invertebrates which are known to be immunogenic and allergenic. We report the identification, isolation and characterisation of the cDNAs of three genes (FucTA, FucTB and FucTC) encoding proteins similar to alpha1,3-fucosyltransferases in Arabidopsis thaliana. Reverse transcription-polymerase chain reaction was used to amplify the full length coding sequence of FucTA. The FucTA gene, which consists of seven exons, encodes a presumptive protein of 501 amino acids showing an overall sequence identity of 66% to the protein encoded by the recently isolated mung bean Fuc-T C3 cDNA. FucTA was expressed in Pichia pastoris under the control of the AOX1 gene promoter. The soluble enzyme was found to catalyse the same reaction as mung bean core alpha1,3-fucosyltransferase as judged by analyses of the products by MALDI-TOF and high-performance liquid chromatography. The FucTB cDNA was isolated from a lambda-ZAP library, but the clone used an alternative splicing site between the second and third exon resulting in a premature stop codon. The FucTC gene encodes a protein with less than 40% identity to FucTA across 115 amino acids of a total of 401 amino acids and is a member of a new sub-family of plant alpha1,3/4-fucosyltransferase homologues.

Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseadish peroxidase epitope.

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core alpha1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm). The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.