A dark and constitutively active mutant of the tiger salamander UV pigment

A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.     

Enhanced antiviral and opsonic activity of a human mannose-binding lectin and surfactant protein D chimera

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.     

The alternative sigma factor sigma is required for resistance of Salmonella enterica serovar Typhimurium to anti-microbial peptides

The enteric pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters a variety of anti-microbial peptides during the course of infection. We report here that the extracytoplasmic sigma factor sigma(E) (RpoE) is required for Salmonella resistance to killing by the bactericidal/permeability-increasing protein (BPI)-derived peptide P2 and the murine alpha-defensin cryptdin-4 (Crp4). Moreover, sigma(E)-deficient S. Typhimurium is attenuated for virulence after oral infection of immunocompromised gp91phox(-/-) mice that lack a functional NADPH phagocyte oxidase, suggesting that sigma(E) plays an important role in resistance to non-oxidative mucosal host defences such as anti-microbial peptides. Although both P2 and Crp4 target the cell envelope, bacterial killing by these peptides appears to occur by distinct mechanisms. Formate enhances bacterial resistance to P2, as previously demonstrated, but not to Crp4. Both sigma(E) and cytoplasmic membrane-associated formate dehydrogenase are required for the protective effect of formate against P2. In contrast to P2, Crp4 does not inhibit bacterial respiration at lethal concentrations. However, both peptides induce expression of rpoE, suggesting that they trigger a common mechanism for sensing extracytoplasmic stress.     

Compensatory role of PspA, a member of the phage shock protein operon, in rpoE mutant Salmonella enterica serovar Typhimurium

Sigma(E) is an alternative sigma factor that responds to and ameliorates extracytoplasmic stress. In Salmonella enterica serovar Typhimurium (S. Typhimurium), sigma(E) is required for oxidative stress resistance, stationary-phase survival and virulence in mice. Microarray analysis of stationary-phase gene expression in rpoE mutant bacteria revealed a dramatic increase in expression of pspA, a member of the phage shock protein (psp) operon. The psp operon can be induced by filamentous bacteriophages or by perturbations of protein secretion, and is believed to facilitate the maintenance of proton motive force (PMF). We hypothesized that increased pspA expression may represent a compensatory response to the loss of sigma(E) function. Increased pspA expression was confirmed in rpoE mutant Salmonella and also observed in a mutant lacking the F(1)F(0) ATPase. Alternatively, expression of pspA could be induced by exposure to CCCP, a protonophore that disrupts PMF. An rpoE pspA double mutant strain was found to have a stationary-phase survival defect more pronounced than that of isogenic strains harbouring single mutations. The double mutant strains were also more susceptible to killing by CCCP or by a bactericidal/permeability-increasing protein (BPI)-derived anti-microbial peptide. Using fluorescence ratio imaging, differences were observed in the Deltapsi of wild-type and rpoE or pspA mutant bacteria. These findings suggest that pspA expression in S. Typhimurium is induced by alterations in PMF and a functional sigma(E) regulon is essential for the maintenance of PMF.     

Role of IL-18 in acute lung inflammation.

We have examined the role of IL-18 after acute lung inflammation in rats caused by intrapulmonary deposition of IgG immune complexes. Constitutive IL-18 mRNA and protein expression (precursor form, 26 kDa) were found in normal rat lung, whereas in inflamed lungs, IL-18 mRNA was up-regulated; in bronchoalveolar (BAL) fluids, the 26-kDa protein form of IL-18 was increased at 2-4 h in inflamed lungs and remained elevated at 24 h, and the "mature" protein form of IL-18 (18 kDa) appeared in BAL fluids 1-8 h after onset of inflammation. ELISA studies confirmed induction of IL-18 in inflamed lungs (in lung homogenates and in BAL fluids). Prominent immunostaining for IL-18 was found in alveolar macrophages from inflamed lungs. When rat lung macrophages, fibroblasts, type II cells, and endothelial cells were cultured in vitro with LPS, only the first two produced IL-18. Intratracheal administration of rat recombinant IL-18 in the lung model caused significant increases in lung vascular permeability and in BAL content of neutrophils and in BAL content of TNF-alpha, IL-1beta, and cytokine-induced neutrophil chemoattractant, whereas intratracheal instillation of anti-IL-18 greatly reduced these changes and prevented increases in BAL content of IFN-gamma. Intratracheal administration of the natural antagonist of IL-18, IL-18 binding protein, resulted in suppressed lung vascular permeability and decreased BAL content of neutrophils, cytokines, and chemokines. These findings suggest that endogenous IL-18 functions as a proinflammatory cytokine in this model of acute lung inflammation, serving as an autocrine activator to bring about expression of other inflammatory mediators.     

Induction of macrophage matrix metalloproteinase biosynthesis by surfactant protein D

Recent studies strongly suggest that surfactant protein D (SP-D) plays important roles in pulmonary host defense and the regulation of immune and inflammatory reactions in the lung. Although SP-D can bind to alveolar macrophages and can elicit their chemotaxis, relatively little is known about the direct cellular consequences of SP-D on the function of these cells. Because matrix metalloproteinases (MMPs) are synthesized in increased amounts in response to various proinflammatory stimuli, we investigated the capacity of SP-D to modulate the production of MMPs by freshly isolated human alveolar macrophages. Unexpectedly we found that recombinant rat SP-D dodecamers selectively induce the biosynthesis of collagenase-1 (MMP-1), stromelysin (MMP-3), and macrophage elastase (MMP-12) without significantly increasing the production of tumor necrosis factor alpha and interleukin-1beta. SP-D did not alter the production of these MMPs by fibroblasts. Phosphatidylinositol, a surfactant-associated ligand that interacts with the carboxyl-terminal neck and carbohydrate recognition domains of SP-D, inhibited the SP-D-dependent increase in MMP biosynthesis. A trimeric, recombinant protein consisting of only the neck and carbohydrate recognition domain did not augment metalloproteinase production, suggesting that the stimulatory effect on MMP production depends on an appropriate spatial presentation of trimeric lectin domains. Although SP-D dodecamers can selectively augment metalloproteinase activity in vitro, this effect may be competitively inhibited by tissue inhibitors of metalloproteinases or surfactant-associated ligands in vivo.     

Cell cycle arrest in cultured neuroblastoma cells exposed to a bis(thiosemicarbazonato) metal complex

Brain tumors such as neuroblastomas and gliomas are often refractory to current treatments. Development of metal-based drugs may offer an alternative approach due to the ability to deliver radionuclides or cytotoxic metals to the tumor. Previous studies have shown that diacetyl-bis(N(4)-methylthiosemicarbazonato)-copper(II) (Cu^II(atsm)) can selectively target hypoxic tumors and this feature has been utilized for development of imaging and radiotherapy. However, we have recently shown that glyoxal-bis(N(4)-methylthiosemicarbazonato)-copper(II) (Cu^II(gtsm)) can target the brain in animal models of neurodegeneration. Unlike Cu^II(atsm), Cu^II(gtsm) is able to release Cu intracellularly under normoxic conditions. Glyoxal-bis(thiosemicarbazones) have reported anticancer effects but little is known about the cellular mechanisms involved. Therefore, in this study, we used protein microarray analysis to investigate the effect of Cu^II(gtsm) on neuroblastoma cell growth in vitro. Treatment of the human neuroblastoma cell line BE(2)-M17, resulted in cell cycle arrest as assessed by fluorescent activated cell sorting (FACS) analysis. Rapidly arrested growth was not associated with onset of apoptosis. Instead, protein microarray analysis revealed that Cu^II(gtsm) rapidly and potently reduced cyclin D1 expression, while increasing Kip2 expression. Other changes observed were decreased Cdk7 expression and activation of CHK2. These changes may be associated with the cell cycle arrest. We also observed a potent decrease of total and phosphorylated insulin-like growth factor receptor (IGF-IR) by Cu^II(gtsm) which is associated with modulation of cyclin D1 expression. Our studies reveal important insights into the potential anticancer activity of Cu^II(gtsm). Further studies are needed to examine the therapeutic potential of Cu^II(gtsm) and other bis(thiosemicarbazonato) metal complexes as metallo-drugs for treatment of systemic or brain tumors.     

Signal transduction from membrane to nucleus: The special case for neurons

Neurons have a unique problem with signal transduction from the membrane in the region of their terminals back to the cell body and nucleus. This distance may be several meters in some nerves in some species, so there is a requirement for some mechanism to stabilize the signal. This review examines two complementary mechanisms for this signal transduction, either by the retrograde axonal transport of the neurotrophic factor together with its receptor, or the transport of a stable activated second messenger molecule. Extrapolation of studies on the fibroblast signal transuction pathway, where it has been shown that G, can translocate from the membrane to the nucleus, has The alpha subunits of both G_i and G_z are retrogradely transported and G_zα or possibly the intact heterotrimeric G_z subsequently accumulates in dorsal root ganglia nuclei. Thus G_z, G_i, and potentially other G-proteins and distinct signaling molecules may provide additional signal transduction pathways to that of the neurotrophins from terminal to nucleus. Special issue dedicated to Dr. Hans Thoenen.