Monomeric rhodium(II) catalysts for the preparation of aziridines and enantioselective formation of cyclopropanes from ethyl diazoacetate at room temperature.

A family of bis(oxazoline) complexes of coordinatively unsaturated monomeric rhodium(II) (2a,b, 3a,b) are described. These complexes serve as catalysts for cyclopropanation of olefins by ethyl diazoacetate, giving excellent yields (66-94%). Enantioselectivities for the cis product isomers are good (61-84%). The reaction shows an unusual preference for formation of the cis isomers. Catalytic aziridination of N-aryl imines with ethyl diazoacetate is also described.

Methimazole as an antioxidant and immunomodulator in thyroid cells: mechanisms involving interferon-gamma signaling and H(2)O(2) scavenging.

The antithyroid drug, methimazole (MMI) is used to treat patients with Graves' hyperthyroidism. The major action of MMI is to inhibit synthesis of thyroid hormone in the thyroid gland. However, MMI also has antioxidant and immunomodulatory effects on thyrocytes and/or immune cells. This study identifies novel antioxidant and immunomodulatory effects of MMI involving the interferon-gamma (IFN-gamma) signaling pathway in thyroid cells. MMI inhibits transcription of the intercellular adhesion molecule-1 (ICAM-1) gene by modulating the function of transcription factor STAT1 (signal transducer and activator of transcription 1), which binds to the IFN-gamma activated site of the ICAM-1 promoter. Furthermore, MMI rapidly eliminates H(2)O(2) produced by IFN-gamma treatment in thyroid cells and thus inhibits the H(2)O(2)-mediated phosphorylation of tyrosine 701 in STAT1. MMI also eliminates H(2)O(2) in vitro. MMI facilitates electron transfer from NADPH to H(2)O(2) using thioredoxin or glutathione, fulfilling a role similar to peroxiredoxin or glutathione peroxidase, respectively. MMI prevents the IFN-gamma and H(2)O(2)-mediated reversible inactivation of phosphatases. These effects inhibit full activation of the IFN-gamma-induced Janus kinase(JAK)/STAT signaling pathway in FRTL-5 thyroid cells. These results may in part explain the antioxidant and immunomodulatory effects of MMI in thyroid cells of Graves' disease patients.

Thyroid-stimulating hormone transcriptionally regulates the thiol-specific antioxidant gene.

Thiol-specific antioxidant (TSA) plays an important role in regulating cell differentiation and proliferation by modulating the hydrogen peroxide (H2O2) mediated responses in a variety of mammalian cells. Thyroid cells are constantly exposed to the actions of reactive oxygen species (ROS), because they produce high levels of H2O2 in response to the physiological action of TSH (thyroid-stimulating hormone). Thyrocytes have several defense mechanisms against ROS, including TSA and SOD (superoxide anion dismutase). Using Northern blot hybridization, we tested the effects of TSH on TSA gene expression in FRTL-5 cells derived from rat thyroids. TSA mRNA expression increased following treatment of cells with TSH at concentrations greater than 10(-9) M. This effect was observed within 6 hours following treatment, and peaked at 8 hours. The effect was blocked by actinomycin D, but not by cycloheximide. The half-life of TSA mRNA was approximately 5.5 hours in the presence or absence of TSH, and that was not affected by TSA mRNA stability. The effects on TSA gene expression were specific to TSH. Other growth factors (e.g., insulin, transferrin and hydrocortisone) did not alter TSA expression. Our results are the first indication that TSH regulates the expression of TSA transcriptionally in thyrocytes.

Cell type-dependent regulation of human DNA topoisomerase III alpha gene expression by upstream stimulatory factor 2.

Here, we report that an E-box element located within the human topoisomerase III alpha (hTOP3 alpha) gene promoter acts as a cell type-specific enhancer. The upstream stimulatory factor (USF) was shown to specifically recognize the mutationally sensitive E-box element. When assayed by transient transfection with hTOP3 alpha promoter-dependent reporter genes, USF is transcriptionally active in HeLa cells but lacks transcriptional activity in Saos-2 cells. The hTOP3 alpha mRNA level in Saos-2 cells was reduced to about 30% of the level observed for HeLa cells, suggesting that the inactivity of USF in hTOP3 alpha promoter activity may be the cause of the marked reduction of hTOP3 alpha mRNA levels in Saos-2 cells. Using transient transfection assays in HeLa cells, we demonstrated that ectopically expressed USF2, but not USF1, was capable of activating hTOP3 alpha transcription through the E-box element. However, USF2 did not stimulate hTOP3 alpha promoter activity in Saos-2 cells. This cell type-specific regulation of promoter activity by USF2 may provide a mechanism for the differential expression of hTOP3 alpha in various tissues and during developmental stages.

Regulation of phosphatidylinositol-phosphate kinase IIgamma gene transcription by thyroid-stimulating hormone in thyroid cells.

This study was performed to evaluate the effects of thyroid-stimulating hormone (TSH) on phosphatidylinositol-4-phosphate 5-kinase type IIgamma (PIPKIIgamma) gene expression in the thyrocytes of FRTL-5 cells. Although PIPKIIgamma mRNA was expressed constantly in the absence of added TSH, its expression increased remarkably in the presence of 10(-9) M TSH. This increase started within 6 h of the addition of TSH, and reached a maximum at 8 h. The mRNA expression properties of PIPKIIgamma in the cells were identified using inhibitors. Actinomycin D blocked PIPKIIgamma transcription strongly, while cycloheximide did not. In an experiment using 5,6-dichlo-1-beta-d -ribofuranosylbenzimidaxole, the half-life of PIPKIIgamma mRNA was approximately 6 h in the presence or absence of TSH, and it was not affected by the stability of the PIPKIIgamma mRNA. The effects of TSH on PIPKIIgamma gene expression were specific, and other growth factors examined (transferrin, insulin and hydrocortisone) did not alter its expression. It is possible that the mechanism of PIPKIIgamma gene expression is involved in the permissive effect of the TSH-cAMP cascade proper. Our results indicate, for the first time, that the expression of PIPKIIgamma is regulated transcriptionally by TSH in thyrocytes.

Heterogeneous nuclear ribonucleoproteins C1 and C2 associate with the RNA component of human telomerase.

Here we demonstrate that heterogeneous nuclear ribonucleoproteins (hnRNPs) C1 and C2 can associate directly with the integral RNA component of mammalian telomerase. The binding site for hnRNPs C1 and C2 maps to a 6-base uridylate tract located directly 5' to the template region in the human telomerase RNA (TR) and a 4-base uridylate tract directly 3' to the template in the mouse TR. Telomerase activity is precipitated with antibodies specific to hnRNPs C1 and C2 from cells expressing wild-type human TR but not a variant of the human TR lacking the hnRNPs C1 and C2 binding site, indicating that hnRNPs C1 and C2 require the 6-base uridylate tract within the human TR to associate with the telomerase holoenzyme. In addition, we demonstrate that binding of hnRNPs C1 and C2 to telomerase correlates with the ability of telomerase to access the telomere. Although correlative, these data do suggest that the binding of hnRNPs C1 and C2 to telomerase may be important for the ability of telomerase to function on telomeres. The C proteins of the hnRNP particle are also capable of colocalizing with telomere binding proteins, suggesting that the C proteins may associate with telomeres in vivo. Therefore, human telomerase is capable of associating with core members of the hnRNP family of RNA binding proteins through a direct and sequence-specific interaction with the human TR. This is also the first account describing the precise mapping of a sequence in the human TR that is required to associate with an auxiliary component of the human telomerase holoenzyme.

Thyrotropin modulates interferon-gamma-mediated intercellular adhesion molecule-1 gene expression by inhibiting Janus kinase-1 and signal transducer and activator of transcription-1 activation in thyroid cells.

TSH is known as an important hormone that plays the major role not only in the maintenance of normal physiology but also in the regulation of immunomodulatory gene expression in thyrocytes. The adhesion molecule intercellular adhesion molecule-1 (ICAM-1) was identified as one of the proteins that are abnormally expressed in the thyroid gland during autoimmune thyroid diseases. In this study we found that TSH inhibits interferon-gamma (IFNgamma)-mediated expression of the ICAM-1 gene, and we investigated the involved mechanisms in rat FRTL-5 thyroid cells. After exposure to IFNgamma, ICAM-1 expression is positively regulated at the level of transcription. This effect occurs via the IFNgamma-activated site (GAS) element in the ICAM-1 promoter as a consequence of the activation of STAT1 (signal transducer and activator of transcription-1), but not of STAT3. On the other hand, after exposure to TSH plus IFNgamma, ICAM-1 transcription is negatively modulated. We found that this inhibitory effect of TSH also occurs via the GAS element. Electrophoretic mobility shift assays confirmed that the IFNgamma-induced DNA-binding activities of STAT1 were reduced by TSH. Furthermore, our results showed that the inhibitory effect of TSH on IFNgamma signaling is caused by inhibition of tyrosine phosphorylation on STAT1, Janus kinase-1 (Jak1), and IFNgamma receptor a, but not Jak2. In conclusion, we have identified a novel mechanism in which TSH modulates the IFNgamma-mediated Jak/STAT signaling pathway through the inhibition of Jak1 and STAT1.

Role of peroxiredoxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells.

Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. One mechanism for this action involves modulation of hydrogen peroxide (H(2)O(2))-mediated cellular responses. This report examines the expression of Prx I and Prx II in thyroid cells and their roles in eliminating H(2)O(2) produced in response to thyrotropin (TSH). Prx I and Prx II are constitutively expressed in FRTL-5 thyroid cells. Prx I expression, but not Prx II expression, is stimulated by exposure to TSH and H(2)O(2). In addition, methimazole induces a high level of Prx I mRNA and protein in these cells. Overexpression of Prx I and Prx II enhances the elimination of H(2)O(2) produced by TSH in FRTL-5 cells. Treatment with 500 micrometer H(2)O(2) causes apoptosis in FRTL-5 cells as evidenced by standard assays of apoptosis (i.e. terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling, BAX expression, and poly(ADP-ribose) polymerase cleavage. Overexpression of Prx I and Prx II reduces the amount of H(2)O(2)-induced apoptosis measured by these assays. These results suggest that Prx I and Prx II are involved in the removal of H(2)O(2) in thyroid cells and can protect these cells from undergoing apoptosis. These proteins are likely to be involved in the normal physiological response to TSH-induced production of H(2)O(2) in thyroid cells.

Involvement of JAK/STAT (Janus kinase/signal transducer and activator of transcription) in the thyrotropin signaling pathway.

TSH is an important physiological regulator of growth and function in thyroid gland. The mechanism of action of TSH depends on interaction with its receptor coupled to heterotrimeric G proteins. We show here that TSH induces the phosphorylation of tyrosine in the intracellular kinases Janus kinase 1 (JAK1) and -2 (JAK2) in rat thyroid cells and in Chinese hamster ovary (CHO) cells transfected with human TSH receptor (TSHR). The JAK family substrates STAT3 (signal transducers and activators of transcription) are rapidly tyrosine phosphorylated in response to TSH. We also find that JAK1, JAK2, and STAT3 coprecipitate with the TSHR, indicating that the TSHR may be able to signal through the intracellular phosphorylation pathway used by the JAK-STAT cascade. TSH increases STAT3-mediated promoter activity and also induces endogenous SOCS-1 (suppressor of cytokine signaling-1) gene expression, a known target gene of STAT3. The expression of a dominant negative form of STAT3 completely inhibited TSH-mediated SOCS-1 expression. These findings suggest that the TSHR is able to signal through JAK/STAT3 pathways.

Thyrotropin induces SOCS-1 (suppressor of cytokine signaling-1) and SOCS-3 in FRTL-5 thyroid cells.

TSH has multiple physiological roles: it is required for growth, differentiation, and function of the thyroid gland, and it regulates transcription of interferon-gamma (IFN-gamma)-responsive genes in thyrocytes, including genes for the major histocompatibility complex and intercellular adhesion molecule-1. This report demonstrates that TSH induces the expression of suppressor of cytokine signaling (SOCS)-1 and -3 proteins and alters the phosphorylation state of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3. The expression of SOCS-1 and SOCS-3 and the phosphorylation state of STAT1 and STAT3 were examined after treatment with TSH or IFN-gamma in either TSH-sensitive FRTL-5 thyroid cells or TSH-insensitive FRT and buffalo rat liver (BRL) cells, which lack functional TSH receptors. SOCS-1 and SOCS-3 are constitutively expressed in FRTL-5 cells, but not in FRT and BRL cells. IFN-gamma up-regulated SOCS-1 and SOCS-3 RNA and protein in FRTL-5 cells, as reported previously for nonthyroid cells. Interestingly, TSH also significantly induced SOCS-1 and SOCS-3 in FRTL-5 cells, but not in FRT and BRL cells. When SOCS-1 or SOCS-3 was overexpressed in FRTL-5 cells, STAT1 phosphorylation at Y701 and STAT1/DNA complex formation in response to IFN-gamma were reduced. Furthermore, overexpression of either SOCS-1 or SOCS-3 significantly inhibited the IFN-gamma-mediated transactivation of the rat ICAM-1 (intercellular adhesion molecule-1) promoter. TSH and IFN-gamma had different effects on STAT1 and STAT3 phosphorylation. The phosphorylation of Y701 in STAT1, which is responsible for homodimer formation, nuclear translocation, and DNA binding, was specifically stimulated by IFN-gamma, but not by TSH or forskolin. However, the phosphorylation of S727 in STAT1 was induced by IFN-gamma, TSH, and forskolin. TSH induced phosphorylation of both Y705 and S727 in STAT3, while IFN-gamma phosphorylated only the Y705. In addition, we found that SOCS-3 was associated with JAK1 and JAK2 and that these associations were stimulated by TSH. These findings demonstrate that TSH induces SOCS in thyroid cells and provides the evidence of signal cross-talk between TSH and cytokines in thyroid cells.

Reduced activity of topoisomerase II in an Adriamycin-resistant human stomach-adenocarcinoma cell line.

A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIalpha or IIbeta showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIalpha and topo IIbeta mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIalpha in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIbeta levels were similar in both lines. Moreover, the amount of in vivo topo IIalpha-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIalpha gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIalpha polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIalpha may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.

Identification of a ribosomal frameshift in Leishmania RNA virus 1-4.

Double-stranded Leishmania RNA virus 1-4 (LRV 1-4) has at least four open reading frames (ORFs). The two small ORFs located near its 5' terminus, ORF1 and ORFx, could encode 34- and 60-amino acid polypeptides, respectively. ORF2 encodes an 82-kDa major capsid protein, and ORF3 encodes a 98-kDa polypeptide which contains the consensus sequence for RNA-dependent RNA polymerases of plus-strand and double-stranded RNA viruses. The complete sequence of LRV 1-4 shows that ORF2 and ORF3 overlap by 71 nucleotides, and that ORF3 lacks a potential translation initiation site, suggesting that the viral polymerase may be synthesized as a 180-kDa fusion protein with the virus capsid. In this report, we present evidence for the synthesis of a fusion protein through a ribosomal frameshift. In vitro-translation experiments and immunostudies involving antiserum against the viral capsid protein demonstrated that the overlapping 71 nucleotides of ORF2 and ORF3 are contained in a region which promotes translational frameshifting. Computer analysis of the putative frameshift region revealed a potential pseudoknot structure located within the overlapping 71 nucleotide sequence.

Sclerosis of hepatic cavernous hemangioma: CT findings and pathologic correlation.

We report a case of hepatic cavernous hemangioma with computed tomographic findings of well demarcated nodular lesser attenuation foci within the main low attenuation mass on precontrast scans and non-enhancement of the foci even on the delayed contrast scans. These have been described as one of the atypical findings of cavernous hemangioma earlier in the literature. Surgery proved that sclerosis accounted for the hypodense nodular densities within the hepatic cavernous hemangioma.

Three-dimensional demonstration and endoscopic treatment of pancreaticoperitoneal fistula.

An alcoholic man presented with bloody ascites, which was confirmed as pancreatic ascites complicating chronic pancreatitis. Endoscopic retrograde pancreatography [ERP] demonstrated a ductal disruption at the head of the pancreas, a fistulous tract, and extravasation to the peritoneal cavity. Furthermore, a computerized tomographic scan subsequent to the endoscopic retrograde pancreatography (ERP-CT scan) gave the three-dimensional anatomy of the fistulous tract by the residual contrast media in the pancreatic duct and the fistulous tract. The pancreatic ascites, which was refractory to conventional medical treatment of a 5-wk duration, was successfully treated by endoscopic placement of a pancreatic stent and administration of a somatostatin analogue.