The serpin-proteinase complex revealed.
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Protein Conformation↗
Biomedical subjects
D A Lawrence
Publications and source records attributed to D A Lawrence.
In utero management of hydrops fetalis caused by critical aortic stenosis.
Hydrops fetalis is rarely associated with congestive heart failure caused by obstructive left-sided heart lesions. There are rare cases of live born neonates with critical congenital valvar aortic stenosis and hydrops reported in the literature, all with fatal outcomes. This report describes, to the best of our knowledge, the first two newborns who were diagnosed prenatally to have hydrops fetalis caused by critical valvar aortic stenosis, who were treated prenatally with digoxin and who postnatally had successful percutaneous balloon aortic valvuloplasty. Both patients had not only left but right ventricular dysfunction. We speculate that right ventricular dysfunction was a contributing factor in the development of hydrops in these patients and in utero medical therapy with digoxin is associated with resolution of the hydrops before delivery.
Angioplasty, Balloon↗
Flow cytometric measurements of neuronal death triggered by PCBs.
We have examined the effects of several individual polychlorinated biphenyl (PCB) congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on viability and intracellular calcium concentration in acutely dissociated cerebellar granule cell neurons obtained from 7-14 day old rat pups, using a flow cytometer. Cell death was determined using the fluorescent dye, 7-aminoactinomycin-D (7-AAD), while intracellular calcium concentration was determined using Fluo-3-AM. When acutely isolated neurons were exposed to 2,4,4' trichlorobiphenyl (2,4,4', there was a dose- and time-dependent loss of viability beginning within 5 min. At a concentration of 100 microM, 80% of neurons were dead within 45 min. Loss of viability was preceded by an apparent increase in cell granularity. No significant cell death was observed upon exposure to the highly dioxin-like coplanar PCB, 3,4,5,3',4', at a concentration of 100 microM or by TCDD at a concentration of 0.1 microM. A slight loss of viability was seen with the ortho 2,6,2',6' and the coplanar 3,4,3',4', but for both it was small. All of the PCB congeners caused an early, transient increase in intracellular free calcium concentration, although TCDD did not. A small, late increase in intracellular free calcium was seen with 2,4,4', probably reflecting events leading to loss of viability. We conclude that at least one PCB congener, 2,4,4', is very neurotoxic and can cause rapid death of cerebellar granule cells.
Animals↗
The serpin PAI-1 inhibits cell migration by blocking integrin alpha V beta 3 binding to vitronectin.
During wound healing, migrating cells increase expression of both the vitronectin receptor (VNR) integrins and plasminogen activators. Here we report that vitronectin significantly enhances the migration of smooth muscle cells (SMCs), and that the specific VNR alpha V beta 3 is required for cell motility. We also show that the alpha V beta 3 attachment site on vitronectin overlaps with the binding site for plasminogen activator inhibitor (PAI)-1, and that the active conformation of PAI-1 blocks SMC migration. This effect requires high-affinity binding to vitronectin, and is not dependent on the ability of PAI-1 to inhibit plasminogen activators. Formation of a complex between PAI-1 and plasminogen activators results in loss of PAI-1 affinity for vitronectin and restores cell migration. These data demonstrate a direct link between plasminogen activators and integrin-mediated cell migration, and show that PAI-1 can control cell-matrix interactions by regulating the accessibility of specific cell-attachment sites. This indicates that the localization of plasminogen activators at sites of focal contact does not initiate a proteolytic cascade leading to generalized matrix destruction, but instead is required to expose cryptic cell-attachment sites necessary for SMC migration.
Animals↗
Hydroquinone resistance in a murine myeloblastic leukemia cell line. Involvement of quinone reductase and glutathione-dependent detoxification in nonclassical multidrug resistance.
A hydroquinone-resistant derivative of the M1 cell line, designated M1HQ, was generated and used to evaluate the biochemical mechanism responsible for resistance to oxidative stress-inducing agents. The hydroquinone concentrations that were cytotoxic to 50 and 90% of the parental M1 cell line in 48 hr were 25 and 90 microM, respectively, whereas exposure to 500 microM hydroquinone did not decrease M1HQ viability significantly. M1HQ cells grew slower than M1 cells and exhibited significantly higher resistance to colchicine, doxorubicin, hydrogen peroxide, 4-hydroperoxycyclophosphamide, and 1,3-bis (2-chloroethyl)-1-nitrosourea but not to benzoquinone, vinblastine, or gamma-radiation. M1HQ cells possessed significantly higher levels of total thiols, glutathione, glutathione peroxidase, glutathione reductase, quinone reductase, and gamma-glutamyl transpeptidase than the parental M1 cell line. Steady-state gamma-glutamylcysteine synthetase mRNA expression also was 1.6-fold higher in M1HQ cells. P-glycoprotein transcripts were detectable in both M1 and M1HQ cells, but were 2-fold higher in M1HQ. Multidrug resistance-associated protein transcripts were not detectable in either M1 or M1HQ. Hydroquinone resistance in M1HQ cells was partially reversible with a combination of inhibitors of quinone reductase, gamma-glutamylcysteine synthetase, glutathione peroxidase, and the multidrug resistance-associated protein, but not with inhibitors of P-glycoprotein, gamma-glutamyl transpeptidase, or glutathione-S-transferase. When treated with [14C]hydroquinone, M1HQ cells did not generate significant hydroquinone-protein adducts but did release an adduct similar to N-acetylcysteinyl-benzoquinone. In contrast, numerous [14C]hydroquinone-protein adducts were produced in M1 cells, while the N-acetylcysteinyl-benzoquinone-like molecule was undetectable. Thus, hydroquinone resistance in M1HQ cells appeared to result from a glutathione-dependent detoxification and export mechanism.
Animals↗
Regulation of HLA-DR and invariant chain expression by human peripheral blood mononuclear cells with lead, interferon-gamma, or interleukin-4.
It has been reported that the expression of major histocompatibility complex (MHC) class II can be regulated by lead (Pb) in murine cells. In both human and mouse, the expression of MHC class II and invariant chain (Ii) can be regulated by cytokines, including interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). Herein we report that in humans, as with IL-4, Pb enhanced MHC class II antigen DR (HLA-DR) surface expression by monocytes and B cells; Ii surface expression by monocytes and B cells was not affected by Pb while it was enhanced by IL-4. IFN-gamma increased HLA-DR and Ii surface expression by monocytes but it decreased HLA-DR and Ii surface expression by B cells. Total cellular HLA-DR expression by peripheral blood mononuclear cells (PBMC) was increased by Pb, IFN-gamma, or IL-4. Total cellular Ii (p33 and p35) expression by PRMC was not affected by Pb or IFN-gamma while it was increased by IL-4. In PBMC, the steady-state mRNA levels of HLA-DR alpha and Ii were not affected by Pb; IFN-gamma increased HLA-DR alpha mRNA expression but not Ii; IL-4 increased both mRNA levels of HLA-DR alpha and Ii. Furthermore, Pb, IFN-gamma, or IL-4 significantly increased the total cellular level of HLA-DR:Ii complexes in PBMC while they had no effect on cell surface HLA-DR:Ii complex expression. Overall, these results suggest that, in vitro, Pb, IFN-gamma, and IL-4 differentially modulate HLA-DR and Ii expression by human PBMC.
Adolescent↗
Plasminogen activator inhibitor-1 and vitronectin promote the cellular clearance of thrombin by low density lipoprotein receptor-related proteins 1 and 2.
Thrombin is a multifunctional protein that has both proteinase and growth factor-like activities. Its regulation is largely mediated by interaction with a host of inhibitors including antithrombin III (ATIII), heparin cofactor II (HCII), alpha2-macroglobulin (alpha2-M), protease nexin I, and plasminogen activator inhibitor-1 (PAI-1). ATIII, HCII, and alpha2-M are all abundant in blood and can inactivate blood-borne thrombin leading to rapid hepatic clearance of the thrombin-inhibitor complex. PAI-1 alone, a poor solution phase inhibitor of thrombin, can efficiently inhibit thrombin in the presence of native vitronectin (VN). In this study, active thrombin was found to be efficiently endocytosed and degraded by cultured pre-type II pneumocyte cells, and both processes could be blocked by polyclonal antibodies to PAI-1. When the relative efficiency of cellular endocytosis of thrombin in complex with a number of inhibitors was examined, 125I-thrombin-PAI-1 complexes were most efficiently cleared compared to 125I-thrombin in complex with the serpins ATIII, HCII, alpha1-proteinase inhibitor, or d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone. Low density lipoprotein receptor-related proteins 1 (LRP) and 2 (gp330/megalin) mediate the endocytosis of thrombin-PAI-1, since antagonists of receptor function such as LRP-1 and LRP-2 antibodies and the 39-kDa receptor-associated protein blocked 125I-thrombin-PAI-1 endocytosis and degradation. The LRP-mediated clearance of exogenously added 125I-thrombin by cultured cells was found to be enhanced 5-fold by inclusion of wild-type PAI-1 but by only 2-fold when a mutant form of PAI-1 that is unable to bind VN was included. This wild-type PAI-1 enhancement of 125I-thrombin clearance was found to occur only in the presence of native VN and not with its conformationally altered form. The results highlight a novel mechanism for cellular clearance of thrombin involving native VN promoting the interaction of thrombin and PAI-1 and the subsequent endocytosis of the complex by LRP-1 or LRP-2. This pathway is potentially important for the regulation of the potent biological activities of thrombin, particularly at sites of vascular injury.
Animals↗
The heavy metal lead modulates the expression of both TNF-alpha and TNF-alpha receptors in lipopolysaccharide-activated human peripheral blood mononuclear cells.
It has been shown that lead (Pb) potentiates lipopolysaccharide (LPS) lethality in animals by increasing the secretion and uptake or reactivity of tumor necrosis factor-alpha (TNF-alpha). Herein we report that PbCl2 increased TNF-alpha secretion from LPS-treated human peripheral blood mononuclear cells (PBMC) in a concentration- and time-dependent manner. PbCl2 also increased total cellular TNF-alpha levels but had no effect on the steady-state levels of TNF-alpha mRNA. PbCl2 decreased membrane-associated TNF-alpha (mTNF-alpha) on LPS-treated monocytes, whereas PbCl2 increased TNF-alpha receptor (TNF-R) p55 surface expression, and had no effect on TNF-R p75 surface expression by LPS-treated monocytes. Overall, the results suggest that PbCl2 increases TNF-alpha expression by posttranscriptional mechanisms in human PBMC, and enhances the reactivity and uptake of TNF-alpha by increasing the surface expression of TNF-R p55.
Base Sequence↗
Surface thiols of human lymphocytes and their changes after in vitro and in vivo activation.
Cellular thiols have long been known to play a role in cell activation and proliferation; however, the differential expression of surface thiols on the lymphoid subsets had not been described. Neither was it known whether alteration of surface thiols occurs after exposure to mitogens or infectious agents. Herein, an impermeant thiol-specific fluorescent probe was employed for flow cytometric analysis of surface thiols. Quantification of surface thiols on resting lymphocytes revealed that some subsets expressed different concentrations of surface thiols (CD19+ > CD8+ > CD4+). Furthermore, surface thiols increased on all subsets by 8 h after mitogenic activation. This increase was blocked by cycloheximide or monensin but not by actinomycin D or inhibition of glutathione synthesis by buthionine sulfoximine. In addition, bacitracin, an inhibitor of protein disulfide isomerase, inhibited the increase in surface thiols and DNA synthesis. Lymphocytes from HIV-infected individuals displayed increased surface thiols on CD19+ and CD4+ cells but not CD8+ cells. Although cellular thiols in general have been believed to play a role in protection against oxidants, signaling associated with cell growth, and apoptosis, there is now evidence that changes in exofacial thiols appear to be involved in some of these critical cell reactivities. Thus, quantitative and possibly qualitative differences in surface thiols correlate with membrane differences between lymphoid subsets and with their differential sensitivities to oxidative stress, which suggests that the mechanisms by which surface thiols are maintained and modified after activation are important cellular functions that need to be further evaluated.
Antigens, CD19↗
Influence of oxygen partial pressure on human and mouse myeloid cell line characteristics.
Mouse and human cells have been reported to have different thiol characteristics (J. M. Messina and D. A. Lawrence, 1992, Int. J. Immunopharmacol. 14, 1221-1234). In addition, when cells are undergoing active growth, they usually have an increased thiol content. In an attempt to determine whether a mouse and a human cell line with similar characteristics can be induced to change their phenotype simply by being cultured at a lower oxygen partial pressure (pO2), the human KG-1 and mouse M1 myeloid cell lines were cultured at 5 and 20% oxygen. It is important to note that 5% O2 is close to the physiological pO2, whereas the percentage of O2 usually employed for most in vitro methods is atmospheric pO2. After long-term culturing at 5% (L cell lines) versus 20% O2 (H cell lines), the thiol content of the original (H) cell lines significantly changed. The amount of total and surface thiols was lower in both L cell lines, but only significantly different on the M1 lines, whereas the glutathione content was significantly lower in the L cell lines of KG-1 and M1. The mouse lines showed the greatest sensitivity to pO2 changes; however, the mouse cell lines were not more sensitive to hydrogen peroxide than the human cell lines even though they possess significantly less total thiols and glutathione. Interestingly, cell lines maintained at lower pO2 (physiological pO2) were more sensitive to hydrogen peroxide than their parental counterparts that were maintained at atmospheric oxygen levels.
Animals↗
Lead differentially modifies cytokine production in vitro and in vivo.
An imbalance between helper T cell type 1 (Th1) and helper T cell type 2 (Th2) activation can result in immunodysregulations leading to impaired cell-mediated immunity with an increased incidence of infectious disease or cancer and/or aberrant humoral immunity that may culminate with an autoimmune disease. Mercury, a heavy-metal toxicant, is known to induce renal autoimmunity characterized by a predominant Th2 response. Lead, another metal toxicant, causes enhanced B cell activities and impairs host resistance to several bacterial and viral infections. In addition, Pb was reported to enhance Th2 proliferation and inhibit Th1 proliferation. The differential effects of Pb on Th subset activation have been further investigated. In vitro IL-4 production by a Th2 clone was significantly increased by the addition of PbCl2, whereas IFN gamma production by a Th1 clone was decreased by the addition of PbCl2. When BALB/c mice were subcutaneously exposed to PbCl2, ex vivo Il-4 production by anti-CD3-stimulated splenic T cells was enhanced, but IFN gamma production was inhibited. Additionally, the plasma IL-4 and IgE levels of Pb-exposed mice were increased, and the plasma IFN gamma levels were significantly lowered in the absence of any additional exogenous antigen. In vitro, ex vivo, and in vivo treatment with HgCl2 produced similar findings. This study is the first report of the preferential activation of a Th2 response by Pb in vivo and suggests that PB, like Hg, may induce autoimmune responses by upsetting the balance between Th1- and Th2-like cells, which could enhance production of antibodies to self antigens.
Animals↗
Mercury (Hg 2+) enhances the depressant effect of kainate on Ca-inactivated potassium current in telencephalic cells derived from chick embryos.
The effect of HgCl2 on kainate (KA)-induced depression of voltage-gated potassium (K+) current in chick embryo telencephalic cells was studied using conventional and nystatin-perforated whole-cell patch-clamp recordings, fluorescence imaging, and flow cytometry techniques. Hg2+ (1 microM) alone did not effect the 4-aminopyridine-(4-AP)-sensitive transient K+ current in immature cells (Embryonic Day 5), but irreversibly potentiated the depressant effect of KA on this K+ current. A 50% potentiation of KA-induced depression of the K+ current was produced by an application of 0.19 microM Hg2+. Application of ionomycin (5 microM) or calcium ionophore A23187 (2 microM) suppressed the K+ current. To test the possibility that the 4-AP-sensitive transient K+ current is a Ca-inactivated current, the effect of intracellular Ca2+ concentration ([Ca2+]i) in the range of 30 nM to 2 microM was determined. The amplitude of the K+ current was sensitive to [Ca2+]i with half-maximal inactivation at 370 nm at +60 mV. The concentration-response curve of the K+ current inhibition by [Ca2+]i was shifted to lower [Ca2+]i and the slope of the curve was reduced in the presence of KA. Hg2+ potentiated these effects of KA. The Ca-dependence of the K+ current was maximal at the 5th embryonic day, declined to the 9th embryonic day, and was absent at the 11th embryonic day. Application of Hg2+ (0.1-1 microM) had no effect on the basal [Ca2+]i of freshly dissociated cells (10th day in ovo) and cells in culture (the 4-day cultures from the telencephalon of 5-day-old embryos), but potentiated KA-induced increase of [Ca2+]i in a Ca-free-EGTA solution in a concentration-dependent manner. Moreover 1 microM Hg2+ delayed and reduced the recovery to basal [Ca2+]i after washout of KA. Exposure to 5-30 microM H2+ caused an irreversible decline of membrane resistance, an increased cell size, and reduced cell granularity and complexity. Intracellular recording of spontaneous neuronal activity and immunocytochemical identification showed that the KA/Hg2+-sensitive Ca-inactivated K+ current exists in early differentiating telencephalic neurons. Because depression of the K+ current by KA and Hg2+ decreases the interspike interval and irreversibly perturbs the frequency code of information in the nervous system, the expression of this current during early neuroembryogenesis may be one of the reasons for the developmental toxicity of inorganic mercury.
Animals↗
Inhibition of human T lymphoblast proliferation by hydroquinone.
Hydroquinone (HQ) is a major metabolite of benzene and is present in large quantities in cigarette tar as a result of the combustion of tobacco leaf pigments. We hypothesize that the immunosuppressive effects of cigarette smoking are due, in part, to the deposition of large quantities of HQ in the lungs. Exposure of primary human T lymphoblasts (HTL) in vitro to 50 microM HQ blocked IL-2-dependent proliferation by > 90% with no loss in viability. Inhibition of DNA synthesis was observed immediately after the addition of HQ to the cells. However, this effect could be reversed up to 6 hr later by simply washing the cells and reculturing them in the absence of HQ. HQ did not significantly alter intracellular glutathione levels up to 24 hr later, and the presence of 50 microM 2-mercaptoethanol or 500 microM dithiothreitol during the treatment did not prevent inhibition of DNA synthesis. HQ did not block binding of 125I-IL-2 to the cells, but inhibited the IL-2-dependent progression of HTL through S phase of the cell cycle. These observations demonstrate that HQ, in concentrations comparable to those found in cigarette tar, is a potent inhibitor of IL-2-dependent T cell proliferation and may therefore help to explain the potent immunosuppressive effects of cigarette smoke on lung T lymphocytes.
Cell Cycle↗
Metal-induced modulation of nitric oxide production in vitro by murine macrophages: lead, nickel, and cobalt utilize different mechanisms.
Macrophages (M phi) can be induced to produce nitric oxide (NO), which has been suggested to be important for macrophages to exercise various functions. We have previously reported that an environmental toxicant, lead (Pb), can significantly inhibit NO production by murine splenic M phis. Herein, eight additional metal ions, gold (Au), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), and zinc (Zn), were assessed. In addition to Pb, Hg and Cd significantly suppressed NO production by cytokine (interferon-gamma and tumor necrosis factor-alpha)-stimulated murine M phis. Au and Cu also were inhibitory, but less than Pb, Hg, and Cd. In contrast, Cr and Zn were not modulatory, and Ni and Co significantly enhanced NO production by cytokine-stimulated M phis. The enhancement by Ni and Co was inhibited by the arginine analog N-monomethylarginine. The metals showed different activating/inhibiting profiles when added to a cell-free (activated M phi lysate) NO-producing-system in which inducible NO synthase (iNOS) is already expressed. Cr, Cu, Pb, and Zn moderately suppressed iNOS, which suggests that they may directly modify enzyme or cofactor activity. Cd, Hg, Mg, Ni, or Co did not produce any significant effect on NO production by the cell-free system. Inhibition of NO production by Pb-exposed M phis was not due to decreased expression of iNOS nor limited to its modest direct inhibition of iNOS; thus, other mechanism(s) must be accountable for the efficient Pb-induced inhibition of NO production by M phi. Ni or Co did induce a substantial increase of iNOS protein. Overall, these observations provide additional insight into the means by which metals via inhibition or enhancement of NO production may be pathogenic, by suppression of defense mechanisms or induction of hypersensitivity, respectively.
Animals↗
Differential in vitro effects of physiological and atmospheric oxygen tension on normal human peripheral blood mononuclear cell proliferation, cytokine and immunoglobulin production.
Since the early work of Mischell & Dutton (Science 153, 1004-1008, 1966), it has been recognized that certain lymphocyte cultures are exquisitely sensitive to the harsh effects of atmospheric oxygen tension. The influence of oxygen partial pressure (pO2) on normal human peripheral blood mononuclear cell (PBMC) phenotype, proliferative ability, cytokine, immunoglobulin production, and redox status was examined by culturing PBMC under ambient oxygen (high pO2) or a more physiological pO2 (5% O2; low pO2). Low pO2 conditions promoted a significant increase in overall viable PBMC number and enhanced Concanavalin A (Con A)- or pokeweed mitogen (PWM)-stimulated PBMC proliferation by approximately 30% and 50%, respectively. No differential pO2 effects were apparent on phytohemagglutinin (PHA)- or staphylococcal enterotoxin B (SEB)-induced proliferation. Both resting and Con A-stimulated lymphocytes incubated for 24 h under high pO2 had a greater baseline carboxy-2',7'-dichlorofluorescin (C-DCF) fluorescence, and were less able to quench the effect of H2O2 treatment compared to lymphocytes cultured under low pO2 conditions. Supernatant gamma-IFN, IL-2, and IL-4 concentrations were elevated 50-65% when PBMC were stimulated with Con A for 24 h under low pO2; however, lipopolysaccharide (LPS)-stimulated IL-1 beta production was reduced by over 75%. PWM-stimulated IgM production by PBMC was significantly reduced in day 7 cultures incubated under low pO2, whereas IgG and IgA production remained relatively unaltered. Immunophenotyping analyses did not reveal any significant alterations in cell subset or marker distribution at the time points examined; however, an interesting trend of increased CD69 expression was observed for Con A-stimulated PBMC incubated under low pO2. These results demonstrate that O2 is a critical parameter for the in vitro culture of lymphocytes, and suggests that varying pO2 may differentially alter PBMC functionality.
Adult↗
Blindness in South Australian kangaroos.
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Animals↗
Genetic mapping of GLCLC, the human gene encoding the catalytic subunit of gamma-glutamyl-cysteine synthetase, to chromosome band 6p12 and characterization of a polymorphic trinucleotide repeat within its 5' untranslated region.
The first and rate-limiting step in the formation of glutathione is catalyzed by gamma-glutamylcysteine synthetase (glutamate-cysteine ligase, E.C. 6.3.2.2). Herein, we describe a trinucleotide repeat polymorphism located in the 5' untranslated region of the human gene, GLCLC, that encodes the catalytic subunit of this enzyme, and we genetically map GLCLC to band 6p12, 1.6 cM distal to D6S295.
Alleles↗
Transforming growth factor-beta: a general review.
Three isoforms of Transforming Growth Factor-beta (TGF-beta 1, beta 2 and beta 3) exist in mammals. They play critical roles in growth regulation and development. Each isoform is encoded by a unique gene on different chromosomes. All three of these growth factors are secreted by most cell types, generally in a latent form, requiring activation before they can exert biological activity. This activation of latent TGF-beta, which may involve plasmin, thrombospondin and possibly acidic microenvironments, appears to be a crucial regulatory step in controlling their effects. The TGF-betas possess three major activities: they inhibit proliferation of most cells, but can stimulate the growth of some mesenchymal cells; they exert immunosuppressive effects; and they enhance the formation of extracellular matrix. Two types of membrane receptors (type I and type II) possessing a serine/threonine kinase activity within their cytoplasmic domains are involved in signal transduction. Inhibition of growth by the TGF-betas stems from a blockage of the cell cycle in late G1 phase. Among the molecular participants concerned in G1-arrest are the Retinoblastoma (Rb) protein and members of the Cyclin/Cyclin-dependent kinase/Cyclin dependent kinase inhibitor families. In the intact organism the TGF-betas are involved in wound repair processes and in starting inflammatory reactions and then in their resolution. The latter effects of the TGF-betas derive in part from their chemotactic attraction of inflammatory cells and of fibroblasts. From gene knockout and from overexpression studies it has been shown that precise regulation of each isoform is essential for survival, at least in the long term. Several clinical applications for certain isoforms have already shown their efficacy and they have been implicated in numerous other pathological situations.
Animals↗