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ABSTRACT: The potent neuropeptide vasodilator, calcitonin gene-related peptide (CGRP), and the vasoactive peptide adrenomedullin (AM)
are structurally related. Evidence from our laboratory has demonstrated that these peptides have potent microvascular actions
of relevance to cardiovascular and inflammatory effects in health and disease. We wish to further investigate the actions
of these peptides through studies in genetically modified mice. We have developed techniques to enable the quantitative analysis
of CGRP and AM responses in the mouse microvasculature. A mouse isolated mesentery system was developed that measures changes
in perfusion pressure used as an index of microvascular relaxation in the precontracted mesenteric microvascular bed. Bolus
injections of CGRP and AM caused dose-dependent decreases in perfusion pressure that were proportional to vascular relaxation.
An in vivo mouse skin assay was also used in which agents were injected intradermally into the dorsal skin. The effects of
these agents was assessed by the extravascular accumulation of intravenously injected 125I-albumin for their ability to potentiate plasma extravasation induced by a mediator of increased microvascular permeability.
CGRP and AM are not directly active in this assay, because it does not directly measure blood flow. However, the vasodilators
acted in a potent and dose-dependent manner to significantly potentiate edema formation. The results demonstrate the potent
activity of CGRP and the activity (although 100- to 300-fold less potent) of AM. Furthermore, the results demonstrate the
increased potency of CGRP in the microvasculature when compared with the structurally distinct peptide VIP and PGE1.
Journal of Molecular Neuroscience 04/2012; 22(1):117-124. · 2.50 Impact Factor
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ABSTRACT: Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.
Traffic 12/2011; 13(3):483-94. · 4.92 Impact Factor
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ABSTRACT: Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca(2+) transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear. We show that host cell entry by T. cruzi mimics a process of plasma membrane injury and repair that involves Ca(2+)-dependent exocytosis of lysosomes, delivery of acid sphingomyelinase (ASM) to the outer leaflet of the plasma membrane, and a rapid form of endocytosis that internalizes membrane lesions. Host cells incubated with T. cruzi trypomastigotes are transiently wounded, show increased levels of endocytosis, and become more susceptible to infection when injured with pore-forming toxins. Inhibition or depletion of lysosomal ASM, which blocks plasma membrane repair, markedly reduces the susceptibility of host cells to T. cruzi invasion. Notably, extracellular addition of sphingomyelinase stimulates host cell endocytosis, enhances T. cruzi invasion, and restores normal invasion levels in ASM-depleted cells. Ceramide, the product of sphingomyelin hydrolysis, is detected in newly formed parasitophorous vacuoles containing trypomastigotes but not in the few parasite-containing vacuoles formed in ASM-depleted cells. Thus, T. cruzi subverts the ASM-dependent ceramide-enriched endosomes that function in plasma membrane repair to infect host cells.
Journal of Experimental Medicine 05/2011; 208(5):909-21. · 13.85 Impact Factor
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ABSTRACT: Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca(2+)-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca(2+). ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.
The Journal of Cell Biology 06/2010; 189(6):1027-38. · 10.26 Impact Factor
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ABSTRACT: Ca(2+) influx through plasma membrane wounds triggers a rapid-repair response that is essential for cell survival. Earlier studies showed that repair requires the exocytosis of intracellular vesicles. Exocytosis was thought to promote resealing by 'patching' the plasma membrane lesion or by facilitating bilayer restoration through reduction in membrane tension. However, cells also rapidly repair lesions created by pore-forming proteins, a form of injury that cannot be resealed solely by exocytosis. Recent studies indicate that, in cells injured by pores or mechanical abrasions, exocytosis is followed by lesion removal through endocytosis. Describing the relationship between wound-induced exocytosis and endocytosis has implications for the understanding of muscular degenerative diseases that are associated with defects in plasma membrane repair.
Trends in cell biology 11/2008; 18(11):552-9. · 12.12 Impact Factor
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ABSTRACT: Ca2+ influx through plasma membrane lesions triggers a rapid repair process that was previously shown to require the exocytosis of lysosomal organelles (Reddy, A., E. Caler, and N. Andrews. 2001. Cell. 106:157-169). However, how exocytosis leads to membrane resealing has remained obscure, particularly for stable lesions caused by pore-forming proteins. In this study, we show that Ca2+-dependent resealing after permeabilization with the bacterial toxin streptolysin O (SLO) requires endocytosis via a novel pathway that removes SLO-containing pores from the plasma membrane. We also find that endocytosis is similarly required to repair lesions formed in mechanically wounded cells. Inhibition of lesion endocytosis (by sterol depletion) inhibits repair, whereas enhancement of endocytosis through disruption of the actin cytoskeleton facilitates resealing. Thus, endocytosis promotes wound resealing by removing lesions from the plasma membrane. These findings provide an important new insight into how cells protect themselves not only from mechanical injury but also from microbial toxins and pore-forming proteins produced by the immune system.
The Journal of Cell Biology 04/2008; 180(5):905-14. · 10.26 Impact Factor
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ABSTRACT: Bacillus anthracis, a spore forming Gram-positive microbe, is the causative agent of anthrax. Although plasmid encoded factors such as lethal toxin (LeTx), edema toxin (EdTx), and gamma-poly-d-glutamic acid (PGA) capsule are known to be required for disease pathogenesis, B. anthracis genes that enable spore invasion, phagosomal escape and macrophage replication are still unknown. To establish transposon mutagenesis as a tool for the characterization of anthrax genes, we employed the mariner-based mini-transposon Bursa aurealis in B. anthracis strain Sterne 7702. B. aurealis carrying an erythromycin resistance cassette and its cognate transposase were delivered by transformation of two plasmids. B. aurealis transposition can be selected for by temperature shift to prevent plasmid replication and by screening colonies for erythromycin resistance. Using inverse polymerase chain reaction, DNA fragments of 129 random erythromycin-resistant transposon mutants were amplified and submitted to DNA sequence analysis. These studies demonstrate that B. aurealis inserts randomly into the genome of B. anthracis and can therefore be employed for finding genes involved in virulence.
Plasmid 08/2006; 56(1):74-7. · 1.52 Impact Factor
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ABSTRACT: The envelope of Escherichia coli is composed of an asymmetric lipid bilayer containing lipopolysaccharide, phospholipid and outer membrane proteins (OMPs). Physical and chemical stresses impact on the integrity of the outer membrane envelope and trigger the sigma(E)-dependent response, whereby E. coli activates the expression of genes that increase its capacity for folding OMPs and synthesizing lipopolysaccharide (LPS). While it has already been appreciated that misfolded OMPs induce the sigma(E) response, a role for LPS in activating this pathway was hitherto unknown. Here we show that ammonium metavandate (NH4VO3) induces multiple changes in E. coli LPS structure and activates the sigma(E)-dependent response without altering OMP. One such NH4VO3-mediated LPS decoration, the CrcA/PagP-catalysed addition of palmitate to lipid A, appeared to be alone sufficient to activate transcription at sigma(E)-dependent promoters. Furthermore, reduced acylation of LPS, caused by htrB or msbB mutations, also resulted in a constitutive expression of the sigma(E) regulon above wild-type levels. Production of these aberrant outer membrane lipids did not noticeably affect the composition or the amount of OMPs. A model is proposed whereby structural intermediates of the LPS biosynthetic pathway or modified LPS molecules may function as signals that activate the sigma(E) response.
Molecular Microbiology 04/2005; 55(5):1403-12. · 5.01 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: The potent neuropeptide vasodilator, calcitonin gene-related peptide (CGRP), and the vasoactive peptide adrenomedullin (AM) are structurally related. Evidence from our laboratory has demonstrated that these peptides have potent microvascular actions of relevance to cardiovascular and inflammatory effects in health and disease. We wish to further investigate the actions of these peptides through studies in genetically modified mice. We have developed techniques to enable the quantitative analysis of CGRP and AM responses in the mouse microvasculature. A mouse isolated mesentery system was developed that measures changes in perfusion pressure used as an index of microvascular relaxation in the precontracted mesenteric microvascular bed. Bolus injections of CGRP and AM caused dose-dependent decreases in perfusion pressure that were proportional to vascular relaxation. An in vivo mouse skin assay was also used in which agents were injected intradermally into the dorsal skin. The effects of these agents was assessed by the extravascular accumulation of intravenously injected 125I-albumin for their ability to potentiate plasma extravasation induced by a mediator of increased microvascular permeability. CGRP and AM are not directly active in this assay, because it does not directly measure blood flow. However, the vasodilators acted in a potent and dose-dependent manner to significantly potentiate edema formation. The results demonstrate the potent activity of CGRP and the activity (although 100- to 300-fold less potent) of AM. Furthermore, the results demonstrate the increased potency of CGRP in the microvasculature when compared with the structurally distinct peptide VIP and PGE1.
Journal of Molecular Neuroscience 02/2004; 22(1-2):117-24. · 2.50 Impact Factor
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ABSTRACT: Esigma(E) RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of Esigma(E) RNA polymerase. It is shown here that the N-terminal portion of sigma(E), residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of sigma(E), a site that is homologous to region 4, the sigma factor binding site for promoter DNA. Mutations that reduce transcription of Esigma(E) RNA polymerase map to sigma(E) residues 178, 181, and 183. Variant sigma(E) proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA. A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of sigma(E) and inhibits the transcription of Esigma(E) RNA polymerase by blocking promoter recognition.
Journal of Biological Chemistry 08/2002; 277(30):27282-7. · 4.77 Impact Factor
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ABSTRACT: Pathogenic yersiniae secrete 14 Yop proteins via the type III pathway. Synthesis of YopQ occurs when the type III machinery is activated by a low-calcium signal, but not when the calcium concentration is above 100 microM. To characterize the mechanism that regulates the expression of yopQ, mutants that permit synthesis of YopQ in the presence of calcium were isolated. Yersiniae bearing deletion mutations in yopN, tyeA, sycN, or yscB synthesized and secreted YopQ in both the presence and the absence of calcium. In contrast, yersiniae with a deletion in yopD or lcrH synthesized YopQ in the presence of calcium but did not secrete the polypeptide. These variants displayed no defect in YopQ secretion under low-calcium conditions, revealing that yopD and lcrH are required for the regulation of yopQ expression. Experiments with transcriptional and translational fusions to the npt reporter gene suggest that yopD and lcrH regulate yopQ expression at a posttranscriptional step. YopD and LcrH form a complex in the bacterial cytosol and bind yopQ mRNA. Models that can account for posttranscriptional regulatory mechanisms of yop expression are discussed.
Journal of Bacteriology 04/2002; 184(5):1287-95. · 3.83 Impact Factor
