Role for copper in transient oxidation and nuclear translocation of MTF-1, but not of NF-kappa B, by the heme-hemopexin transport system

Heme-hemopexin (2-10 microM) is used as a model for intravenous heme released in trauma, stroke, and ischemia-reperfusion. A transient increase in cellular protein oxidation occurs during receptor-mediated heme transport from hemopexin which is inhibited by the nonpermeable Cu(I) chelator, bathocuproinedisulfonate. Thus, participation of surface redox process involving Cu(I) generation are proposed to be linked to the induction of the protective proteins heme oxygenase-1 (HO-1) and metallothionein-1 (MT-1) by heme-hemopexin. The region (-153 to -42) in the proximal promoter of the mouse MT-1 gene responds to heme- and CoPP-hemopexin in transient transfection assays and contains metal-responsive elements for MTF-1 and an antioxidant-responsive element (ARE) overlapping a GC-rich E-box to which USF-1 and -2 bind. No decreases in DNA binding of the diamide-oxidation sensitive USF-1 and -2 occur upon exposure of cells to heme-hemopexin. MTF-1 and the ARE-binding proteins are relatively resistant to diamide oxidation and are induced approximately eight- and two-fold, respectively, by heme-hemopexin. BCDS prevents the nuclear translocation of MTF-1 by both heme- and CoPP-hemopexin complexes as well as MT-1 mRNA induction by CoPP-hemopexin. Thus, copper is needed for the surface oxidation events and yet the nuclear translocation of MTF-1 in response to hemopexin occurs via copper, probably Cu(I),-dependent signaling cascades from the hemopexin receptor rather than the oxidation per se.     

Role of actin microfilament in osmotic stretch-induced increase of voltage-operated calcium channel current in guinea-pig gastric myocytes

 Using the whole-cell patch clamp technique, the role of actin microfilament in hyposmotic increase of voltage-operated calcium channel current (I _Ba) was studied in guinea-pig gastric myocytes. Hyposmotic superfusate (212 mOsm) increased peak I _Ba amplitude by 32.7 ± 6.5%; when cytochalasin-D (Cyt-D, 20 μM), an actin cytoskeleton disruptor, was used, an increase of only 9.7 ± 3.1% was seen. I _Baresponse to osmotic stress was potentiated (45.1 ± 4.1% increase) by 20 μM phalloidin, an actin microfilament stabilizer. However, colchicine (100 μM), an microtubule cytoskeleton disruptor, had no effect on either I _Ba or its response to hyposmotic solution. Phalloidin also induced a rightward shift of the I/V relationship of I _Ba, while Cyt-D itself had no effect. These results suggest that actin cytoskeleton may mediate hyposmotic stretch-induced I _Ba increase in gastric smooth muscle. Received: 26 March 1997 / Received after revision: 28 May 1997 / Accepted: 3 June 1997     

IFNgamma secretion following stimulation with total tumor peptides from autologous human tumors

Several issues remain to be resolved before the efficacy of various approaches to elicit anti-tumor immunity in patients can be evaluated. First, in vitro assays able to detect responses by T cells primed in vivo are needed. Second, a source of tumor antigen to stimulate patients' lymphocytes in vitro is required. The ELISPOT assay is attractive, because it can be performed with a small numbers of cells and requires only short-term culture in vitro. A source of tumor antigen is more problematic, since for most tumors, tumor-associated antigens (TAA) have not been identified and/or cloned. In this report we demonstrate that autologous antigen-presenting cells (APC) pulsed with total tumor peptides from autologous tumor tissue can stimulate IFNgamma release by patients' lymphocytes in the ELISPOT assay. Thus, this approach should be considered for monitoring immune responses in clinical immunotherapy trials.     

Cell-surface events for metallothionein-1 and heme oxygenase-1 regulation by the hemopexin-heme transport system

A model has been developed for the hemopexin receptor-mediated heme transport system based on iron uptake in yeast. Two steps are required: reduction followed by oxidation by a multi-copper-oxidase. Furthermore, in the hemopexin system, the surface redox events have been linked with gene regulation. The impermeable Cu(I) chelator bathocuproinedisulfonate (BCDS) is shown here to abrogate heme oxygenase-1 (HO-1) mRNA induction by heme-hemopexin. A role for Cu(I) in the regulation of HO-1 and MT-1 (Sung et al., 1999) by hemopexin supports the participation of electron transport processes at the cell surface as does competition by the reductase activator, ferric citrate, which inhibits the induction of MT-1 and HO-1 mRNA by heme-hemopexin. There is a key role for the hemopexin receptor because neither ferric citrate nor iron-transferrin alone regulates MT-1 or HO-1. Cell-surface copper is the first molecule to link the concomitant regulation of HO-1 and MT-1 by the hemopexin receptor. In addition, cytochrome b5 and cytochrome b5 reductase are implicated here in the response of cells to heme-hemopexin. Reduction of one or more electron donors of the reductase and oxidation of the electron acceptor, b5 heme, leads to gene regulation, but only when heme-hemopexin is bound to its receptor. Protein kinase cascades, including JNK, are activated by the hemopexin receptor itself upon ligand binding but are modulated by a Cu(I)-dependent process likely to be heme uptake.     

Multifunctional initiation of lactide polymerization by stannous octoate/pentaerythritol

L -Lactide was polymerized with stannous 2-ethylhexanoate (stannous octoate) in the presence of pentaerythritol to investigate multifunctional initiation. The prepared oligomers contain starshaped 4-arm molecules when the mole ratio of [lactide]/[pentaerythritol] is above 32. The molecular weight of oligomers coincides with the [lactide]/[pentaerythritol] ratio, indicating that pentaerythritol in conjugation with stannous octoate is an initiator for the “living” polymerization of L-lactide.     

ASC:SIL ratio following implementation of the 2001 Bethesda System

The 2001 Bethesda system (TBS 2001) eliminated the "satisfactory but limited by" category, benign cellular changes (BCC), and the designations "favor benign" (ASC-B) and "favor low grade" (ASC-L) for atypical squamous cells. We compared the unsatisfactory rate and atypical squamous cells:squamous intraepithelial lesions (ASC:SIL) ratio pre- and postimplementation of TBS 2001 to see if there was an increase in unsatisfactory specimens, ASC rate, and altered ASC:SIL ratio. Pap Tests (569,726) reviewed at the Cytopathology Laboratory of Women and Infants Hospital from 1998-2002 were included. TBS 1991 terminology was used through December 31, 2001. Conversion to TBS 2001 took place on January 1, 2002. The average ASC:SIL ratios pre- and postimplementation of TBS 2001 were 1.52:1 and 1.42:1, respectively. The rates of unsatisfactory specimens and ASC remained unchanged. Conversion to TBS 2001 did not adversely affect the ASC:SIL ratio or the detection rates of abnormalities of Pap tests.     

Studies on the mechanism of tachyphylaxis to disodium cromoglycate.

The tachyphylaxis to disodium cromoglycate's (DSCG) inhibition of antigen-induced histamine release is readily demonstrable utilizing passively sensitized rat lung fragments. This tachyphylaxis to DSCG is evident whether or not calcium is present during drug preincubation. An attempt to relate the mechanism of tachyphylaxis to the DSCG-induced release of an endogenous cellular inhibitory material was unsuccessful insofar as could be demonstrated by an effect on mediator release.     

Studies on the development of enzyme linked immunosorbent assay (ELISA) for hepatitis B surface antigen(HBsAg) by monoclonal antibodies of different affinity constants

Mouse monoclonal antibodies to Hepatitis B surface antigen(HBsAg) were prepared and their functional capabilities tested by the method of solid phase enzyme linked immuno sorbent assay(ELISA). HBsAg binding studies indicated that one monoclonal antibody 6E-1-1 bound more HBsAg at a faster rate than the other monoclonal antibodies. Also, for the binding inhibition studies with the selected monoclonal antibody 6E-1-1, one monoclonal antibody 8D-3-6 didn’t exhibit binding inhibition for HBsAg. Then, a simultaneous ELISA method was developed for the immunodiagnosis of HBsAg. Different combinations of two monoclonal antibodies as solid phase and horseradish peroxidase(HRPO) labeled phase were studied. The combination of monoclonal antibody of higher affinity constant (6E-1-1) immobilized in a solid phase and monoclonal antibody of lower affinity constant (8D-3-6) as a HRPO labeled phase was more sensitive when two monoclonal antibodies of different affinity constants for HBsAg were prepared.