Non-protein thiols flux to S-nitrosothiols in endothelial cells: an LPS redox signal.

Lipopolysaccharide (LPS) injures blood vessels by activating pathways in the endothelium that lead either to cell survival and proliferation or apoptosis. It has been suggested that these outcomes are determined when reactive oxygen and nitrogen intermediates oxidize low molecular weight non-protein thiols (NPSHs) such as glutathione (GSH) and cysteine (Cys), which serve as major intracellular reducing agents. The oxidoreduction of NPSHs could be an important redox signal if it were shown to occur rapidly following injury. Towards that end, cultured bovine aortic endothelial cells were stained with the thiol fluorescent probe, monobromobimane (MBB). Most of the acid extractable MBB-reactive adducts are GSH (approximately 90%) and Cys (approximately 90%). Within 1 min of LPS exposure, 50-70% of the MBB-reactive NPSHs are consumed without evidence for concomitant net generation of superoxide, hydrogen peroxide, singlet oxygen, or glutathione disulfide (GSSG). Although LPS induces an increased rate of thiol-disulfide exchange, the slight increase does not explain the magnitude of NPSH consumption. Within the first 10 min of recovery from LPS exposure, the MBB-reactive NPSH fluorescence returns at or slightly above baseline values. When HgCl2 was added to the acid extract, one mole of S-nitrosothiol oxidizing equivalent was found for every mole of MBB-reactive NPSH consumed. It is suspected that the rapid flux of MBB-reactive NPSHs and Hg2+-inducible oxidants reflects transition of GSH to GSNO (S-nitrosoglutathione) and could be an important redox signal in endothelial cells exposed to LPS.

Medical considerations for wilderness and adventure travelers.

Wilderness and adventure travel has attracted a growing number of participants. Many adventure travelers are inadequately prepared for their trip and are naive about the associated risks. Physicians are frequently asked to provide medical clearance and to complete medical release forms to travelers. To provide better advice, the physician should be familiar with the rigors and difficulty of these activities and must consider the potential environment hazards and remoteness of the intended area of travel. This article provides information on trip rating scales, environmental hazards, altitude illness, hypothermia, heat illness, and adventure travel medical kits.

H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappaB to the interleukin-8 promoter in endothelial and epithelial cells.

We previously demonstrated that tumor necrosis factor-alpha (TNFalpha) and H2O2 differentially regulate interleukin-8 (IL-8) and intercellular adhesion molecule (ICAM-1) gene expression in endothelial and epithelial cells. H2O2 induced IL-8 expression in the A549 and BEAS-2B epithelial cell lines, but not in the human microvessel endothelial cell line, HMEC-1 or human umbilical vein endothelial cells. In contrast, H2O2 induced ICAM-1 only in endothelial cells. Unlike H2O2, the proinflammatory cytokine TNFalpha induced IL-8 and ICAM-1 in both cell types. In this study, we examine the role of the redox-responsive transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB) in the differential expression of IL-8. DNA binding studies using nuclear protein extracts from HMEC-1 and A549 cells stimulated with H2O2 or TNFalpha demonstrated differential activation and promoter binding of AP-1 and NF-kappaB. H2O2 activated AP-1 but not NF-kappaB in A549, whereas TNFalpha activated AP-1 as well as NF-kappaB. In HMEC-1, TNFalpha activated NF-kappaB but not AP-1, while H2O2 did not activate either transcription factor. The differential activation of the factors was also reflected in their differential binding to the IL-8 promoter. Moreover, the H2O2 concentration dependent increase in epithelial IL-8 mRNA expression directly corresponded to the H2O2 concentration dependent binding of AP-1 to the IL-8 promoter. Supershift analysis revealed H2O2 as well as TNFalpha induced AP-1 complexes containing c-Fos and JunD. TNFalpha induced NF-kappaB complexes containing Rel A (p65). Immunohistochemical staining of HMEC-1 and A549 cells revealed TNFalpha stimulated nuclear localization of Rel A, whereas no translocation of Rel A was detected in either cell type stimulated by H2O2. These data indicate that the cell type-specific induction of IL-8 gene expression by H2O2 and TNFalpha in HMEC-1 and A549 cells can be explained by the differential binding of AP-1 and NF-kappaB to the IL-8 promoter.

Aminothiols protect endothelial cell proliferation against inhibition by lipopolysaccharide.

Lipopolysaccharide (LPS) is a primary agent of sepsis that damages the vascular endothelium. Endothelial cell proliferation is key to the repair of damaged endothelium, and drugs that counteract the antiproliferative impact of LPS on endothelial cells should be beneficial. Because LPS exerts much of its cytotoxicity by generating reactive oxygen and nitrogen intermediates, it would be helpful to know whether therapeutic antioxidant thiols maintain cell proliferation in injured endothelium. In this study, it was found that LPS inhibited bovine aortic endothelial cell proliferation by inducing apoptosis and by decreasing DNA synthesis. Because of its benefit to irradiated endothelial cells, we then treated the cells with a radio- and chemoprotective aminothiol, WR-1065 ([N-2-mecaptoethyl]-1-3-diaminopropane, the active form of Amifostine/Ethyol). WR-1065 attenuated the inhibition of DNA synthesis caused by LPS exposure. The disulfide of WR-1065, WR-33278, was tested and shown to both promote DNA synthesis and inhibit apoptosis. The effectiveness of the disulfide suggests that the reduction of cytotoxicity does not necessarily result from the scavenging of free radicals. These findings demonstrate a novel role for aminothiols in promoting DNA synthesis and lowering apoptosis in endothelium injured with LPS.

Aminothiol WR-1065 protects endothelial cell morphology against alterations induced by lipopolysaccharide.

In septic patients, lipopolysaccharide (LPS) damages the vascular endothelium, which manifests as tissue edema and impaired healing. This pathology occurs when LPS distorts endothelial cell morphology partly by generating free radicals. A radioprotector that scavenges free radicals, the aminothiol WR-1065 ([N-2-mercaptoethyl]-1-3-diaminopropane) was found in a prior study to normalize the morphology of irradiated endothelial cells (Mooteri SN, Podolski JL, Drab EA, et al: Radiat Res 145:217-224, 1996). The aim of this study was to determine whether WR-1065 also normalized endothelial cell morphology following exposure to LPS. For this aim, portions of bovine aortic endothelial cell cultures were denuded and exposed to LPS at 1 ng/mL. After 30 min, the apical membrane expressed increased integrin receptor to fibronectin, alpha5beta1. After 5 h, the morphology of the cells at the leading edge was distorted, and cell-cell contact was lessened. Also, filamentous actin-containing stress fibers were dissipated; however, filamentous actin content per cell was unchanged. Treatment with 2 mM WR-1065 for 2 h prior to LPS exposure attenuated the increased expression of alpha5beta1 and promoted cell-cell contact in the migrating endothelial cells. WR-1065 also promoted the retention of stress fibers and actin cytoskeletal shape in cells treated with LPS. Thus, LPS distorted endothelial cell morphology after increasing apical membrane expression of alpha5beta1 and dissipating stress fibers, effects prevented by WR-1065.

Identification of an activity in B-cell extracts that selectively impairs the formation of an immunoglobulin mu s poly(A) site processing complex.

The immunoglobulin mu heavy-chain transcription unit is differentially expressed during B-cell development, producing mRNAs that encode secreted (mu s) and membrane-bound (mu m) forms of the heavy-chain polypeptide. Whereas the mu s mRNA and the mu m mRNA are produced in approximately equal abundance in B cells, an increase in the utilization of the mu s poly(A) site contributes to the production of the mu s mRNA as the predominant form in a plasma cell. Previous experiments have demonstrated a correlation between the formation of a stable complex on a poly(A) site and the relative function of the poly(A) site. We have thus investigated the parameters determining the interaction of these factors with the immunoglobulin poly(A) sites. Assays of complex formation involving the two immunoglobulin poly(A) sites by using HeLa cell activities revealed the formation of stable complexes with no apparent difference between the mu s site and the mu m site. In contrast, the mu s-specific complex was markedly less stable when a B-cell extract was used. Fractionation of B-cell extracts has revealed an activity that specifically destabilizes the mu s polyadenylation complex, suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors.

Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection.

Analysis of immunoglobulin heavy chain delta transcription termination in the production of delta S or delta M mRNA.

mRNA encoding secreted immunoglobulin is synthesized either by termination of transcription 3' to secreted terminus sequences and 5' to the membrane terminus sequences or by cleavage of a pre-mRNA transcript containing both secreted and membrane sequences at the appropriate polyadenylation site 5' to the membrane sequences. In vitro "run-on" transcription analysis was used to examine the delta transcription termination patterns in resting membrane IgD expressing B lymphocytes, in KWD2, an IgD-secreting hybridoma, and in TEPC 1017, an IgD-secreting plasmacytoma. In resting B cells, transcription terminated in a region 4 to 7 kilobases 3' to the delta M exons. Transcription in the secreting cells continued through the delta M exons, but terminated at more upstream sites. Additionally, an increased loading of polymerases in the region of the delta S exon and its 5' flanking sequence was detected in the secreting cells and was particularly pronounced in TEPC 1017. It is hypothesized that this peak correlates with high delta S mRNA production.

Poly(A) site efficiency reflects the stability of complex formation involving the downstream element.

A critical step in mRNA biogenesis is the generation of the mRNA 3' end through an endonucleolytic cleavage of the primary transcript followed by the addition of a approximately 200 nucleotide (nt) poly(A) tail. The efficiency of poly(A) site function can vary widely and for those genes with multiple poly(A) sites, the choice can be a regulated event. A functional poly(A) site is characterized by cis-acting RNA sequences including the well-conserved AAUAAA hexamer, located 10-30 nt upstream of the cleavage site, and a highly variable downstream GU- or U-rich element. The gene specific nature of the downstream sequence suggests that it may be a primary determinant of poly(A) site efficiency. Several recent studies have detailed the purification of factors that mediate the cleavage and polyadenylation reaction and that recognize the cis-acting signals. Two of these factors are responsible for the formation of a stable, committed ternary complex with the pre-RNA. In order to define the role of this stable complex in poly(A) site function, we have compared the processing efficiency of several pre-mRNAs with the stability of the complex that forms on these RNAs. We show that ternary complex stability reflects both the in vivo and the in vitro efficiency of the poly(A) site and that the stability of this complex is dependent on the nature of the downstream sequence element. We conclude that the stability of these protein--RNA interactions, dictated by the downstream element, plays a major role in determining the processing efficiency of a particular poly(A) site.

Role of transcriptional termination in the regulation of mu mRNA expression in B lymphocytes.

The synthesis of mRNA for mu-chain of membrane vs secretory IgM in normal resting B lymphocytes is stringently regulated. Very minimal amounts of microseconds mRNA are produced until the cells are stimulated. We have found a strong correlation between the initiation of mu S mRNA production and an alteration in the site of termination of polymerases transcribing the mu-gene. Furthermore, since the abundance of mu S mRNA is dependent on the differentiation stage of the B lymphocyte, we examined subpopulations of cells at various stages of activation and found that along with increased initiation of polymerases at the mu-locus, termination is moved further upstream in more highly differentiated cells. Therefore regulation of mu S mRNA may require factor(s) that induce transcriptional termination and these may be related to the factor(s) that augment transcriptional initiation.

The C mu gene is transcribed in IgG bearing B lymphocytes.

During the maturation of an immune response, some IgM bearing B lymphocytes differentiate into IgG secreting cells. Similarly, a subset of normal B cells cultured in the presence of a mitogen will develop into IgG synthesizing cells, many of which continue to express IgM. The experiments described in this paper utilize such IgG3 expressing B cells present in mitogen-stimulated cultures to address the continuing controversy over the molecular mechanism(s) allowing simultaneous expression of IgM and isotypes other than IgD. Nascent transcripts were labelled in vitro in order to determine whether the C mu gene was still transcribed in these cells. If no mu transcription was detected, then this would suggest that the mu chain protein, in cells expressing both IgM and IgG3, must be derived from translation of long-lived mu mRNA. Alternatively, detection of continued mu gene transcription would provide direct evidence for alternate processing of pre-mRNA transcripts encoding both mu and gamma gene sequences. The membrane IgG3+ (mIgG3+) cells were isolated from mitogen-stimulated cultures by staining and then sorting on a Fluorescence Activated Cell Sorter (FACS). Prior to examining mu gene transcription in these cells, it was necessary to ascertain the purity of the sorted population. Accordingly, restaining of the sorted IgG3+ cells showed only a minor contamination by mIgM+IgG3- cells. Additionally, the majority of the mIgG3+ cells were also mIgM+. Most importantly, it was then demonstrated that the IgG3 detected on the cells was intrinsic to them rather than cytophilically adsorbed. By biosynthetic labelling, the sorted IgG3+ population was found to also actively secrete both IgM and IgG3. Examination of transcription showed that the IgG3+ cells were indeed transcribing the mu exons and were doing so at about 50% the level of an equal number of unseparated cells. It was determined that this level of C mu transcription is significantly higher than that which could be derived from the contaminating cells. Thus, it is clear that at least some IgG3 expressing cells have not deleted the C mu gene and therefore the gamma 3 mRNA in a portion of the cells expressing both IgM and IgG3 must be derived from pre-mRNA transcripts which contain both C mu and C gamma 3 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of the gamma 1 gene by lipopolysaccharide and T cell-derived lymphokines containing a B cell differentiation factor for IgG1 (BCDF gamma).

By using both pulse labeling of nascent RNA chains and lactoperoxidase-catalyzed cell surface radioiodination, we examined both the de novo synthesis of mRNA for gamma-chains and the expression of membrane IgG (mIgG) on cells which had been stimulated with LPS plus a T cell supernatant (SN) containing a B cell differentiation factor for IgG1 (BCDF gamma). Our results show that neither nascent mRNA for gamma 1 chains nor mIgG1 can be detected in B lymphocytes until they have been stimulated by both LPS and BCDF gamma-containing T cell SN, and suggest that cell surface expression and secretion of IgG1 are coordinately controlled.