The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

Polynitrosylated proteins: characterization, bioactivity, and functional consequences.

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.

Bioactive and nuclease-resistant l-DNA ligand of vasopressin

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II

Superoxide anion (O2−) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O2− is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O2− production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O2−-generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22phox, gp91phox, p47phox, and p67phox localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O2− production. Furthermore, immunodepletion of p67phox from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67phox. Further study into the regulation of this adventitial source of O2− is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.

Parathyroid hormone-related peptide (PTHrP) regulates fetal–placental calcium transport through a receptor distinct from the PTH/PTHrP receptor

To determine the role of PTHrP in fetal calcium metabolism, blood calcium was measured in mice homozygous (HOM) for deletion of the PTHrP gene. On day 18.5 of gestation, ionized calcium and the maternal–fetal calcium gradient were significantly reduced in HOM PTHrP-ablated fetuses compared with that of their littermates. To assess the placental contribution to the effect of PTHrP, 45Ca and 51Cr-EDTA (as a blood diffusional marker) were administered by intracardiac injection to pregnant, heterozygous dams on day 17.5 of gestation. Five minutes after the injection, whole fetal 45Ca accumulation was significantly decreased in HOM PTHrP-ablated fetuses compared with that of their littermates. Next, two fetuses from each litter were injected in utero with fragments of PTHrP, PTH, or diluent 1 h before administering 45Ca and 51Cr to the dam. PTHrP-(1–86) and PTHrP-(67–86) significantly increased relative 45Ca accumulation in HOM PTHrP-ablated fetuses, but PTHrP(1–34), PTH-(1–84), and the diluent had no effect. Finally, similar studies were performed on fetal mice that lacked the PTH/PTHrP receptor gene. Ionized calcium was significantly reduced in HOM PTH/PTHrP receptor-ablated fetuses. However, 5 min after maternal injection of 45Ca and 51Cr, relative accumulation of 45Ca was significantly increased in these fetuses. It was concluded that PTHrP is an important regulator of fetal blood calcium and placental calcium transport. In addition, the bioactivity of PTHrP for placental calcium transport is specified by a mid-molecular region that does not use the PTH/PTHrP receptor.

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: A possible link between inflammatory cytokines and atherogenesis

Inflammation plays a critical role in atherogenesis, yet the mediators linking inflammation to specific atherogenic processes remain to be elucidated. One such mediator may be secretory sphingomyelinase (S-SMase), a product of the acid sphingomyelinase gene. The secretion of S-SMase by cultured endothelial cells is induced by inflammatory cytokines, and in vivo data have implicated S-SMase in subendothelial lipoprotein aggregation, macrophage foam cell formation, and possibly other atherogenic processes. Thus, the goal of this study was to seek evidence for S-SMase regulation in vivo during a physiologically relevant inflammatory response. First, wild-type mice were injected with saline or lipopolysaccharide (LPS) as a model of acute systemic inflammation. Serum S-SMase activity 3 h postinjection was increased 2- to 2.5-fold by LPS (P < 0.01). To determine the role of IL-1 in the LPS response, we used IL-1 converting enzyme knockout mice, which exhibit deficient IL-1 bioactivity. The level of serum S-SMase activity in LPS-injected IL-1 converting enzyme knockout mice was ≈35% less than that in identically treated wild-type mice (P < 0.01). In LPS-injected IL-1-receptor antagonist knockout mice, which have an enhanced response to IL-1, serum S-SMase activity was increased 1.8-fold compared with LPS-injected wild-type mice (P < 0.01). Finally, when wild-type mice were injected directly with IL-1β, tumor necrosis factor α, or both, serum S-SMase activity increased 1.6-, 2.3-, and 2.9-fold, respectively (P < 0.01). These data show regulation of S-SMase activity in vivo and they raise the possibility that local stimulation of S-SMase may contribute to the effects of inflammatory cytokines in atherosclerosis.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

S-nitrosothiol repletion by an inhaled gas regulates pulmonary function

NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O2-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O2/O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document}. This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O2 or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.

Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation

12/15-Lipoxygenase (LOX) activity is elevated in vascular diseases associated with impaired nitric oxide (⋅NO) bioactivity, such as hypertension and atherosclerosis. In this study, primary porcine monocytes expressing 12/15-LOX, rat A10 smooth muscle cells transfected with murine 12/15-LOX, and purified porcine 12/15-LOX all consumed ⋅NO in the presence of lipid substrate. Suppression of LOX diene conjugation by ⋅NO was also found, although the lipid product profile was unchanged. ⋅NO consumption by porcine monocytes was inhibited by the LOX inhibitor, eicosatetraynoic acid. Rates of arachidonate (AA)- or linoleate (LA)-dependent ⋅NO depletion by porcine monocytes (2.68 ± 0.03 nmol ⋅ min−1 ⋅ 106 cells−1 and 1.5 ± 0.25 nmol ⋅ min−1 ⋅ 106 cells−1, respectively) were several-fold greater than rates of ⋅NO generation by cytokine-activated macrophages (0.1–0.2 nmol ⋅ min−1 ⋅ 106 cells−1) and LA-dependent ⋅NO consumption by primary porcine monocytes inhibited ⋅NO activation of soluble guanylate cyclase. These data indicate that catalytic ⋅NO consumption by 12/15-LOX modulates monocyte ⋅NO signaling and suggest that LOXs may contribute to vascular dysfunction not only by the bioactivity of their lipid products, but also by serving as catalytic sinks for ⋅NO in the vasculature.

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.

The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.

In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the D1 gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, Ga20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H]GA5 into seedlings of d1 and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index. For the controls, GA20 was metabolized to GA1,GA3, and GA5; GA5 was metabolized to GA3. For the d1 mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using d1 seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.