Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

Resistin-Like Molecule α in Allergen-Induced Pulmonary Vascular Remodeling.

Resistin-like molecule α (RELMα) has mitogenic, angiogenic, vasoconstrictive, and chemokine-like properties and is highly relevant in lung pathology. Here, we used RELMα knockout (Retnla(-/-)) mice to investigate the role of RELMα in pulmonary vascular remodeling after intermittent ovalbumin (OVA) challenge. We compared saline- and OVA-exposed wild-type (WT) mice and found that OVA induced significant increases in right ventricular systolic pressure, cardiac hypertrophy, pulmonary vascular remodeling of intra-alveolar arteries, goblet cell hyperplasia in airway epithelium, and intensive lung inflammation, especially perivascular inflammation. Genetic ablation of Retnla prevented the OVA-induced increase in pulmonary pressure and cardiac hypertrophy seen in WT mice. Histological analysis showed that Retnla(-/-) mice exhibited less vessel muscularization, less perivascular inflammation, reduced medial thickness of intra-alveolar vessels, and fewer goblet cells in upper airway epithelium (250-600 µm) than did WT animals after OVA challenge. Gene expression profiles showed that genes associated with vascular remodeling, including those related to muscle protein, contractile fibers, and actin cytoskeleton, were expressed at a lower level in OVA-challenged Retnla(-/-) mice than in similarly treated WT mice. In addition, bronchoalveolar lavage from OVA-challenged Retnla(-/-) mice had lower levels of cytokines, such as IL-1ß, -1 receptor antagonist, and -16, chemokine (C-X-C motif) ligand 1, -2, -9, -10, and -13, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, TIMP metallopeptidase inhibitor-1, and triggering receptor expressed on myeloid cells-1, than did that from WT mice when analyzed by cytokine array dot blots. Retnla knockout inhibited the OVA-induced T helper 17 response but not the T helper 2 response. Altogether, our results suggest that RELMα is involved in immune response-induced pulmonary vascular remodeling and the associated increase in inflammation typically observed after OVA challenge.

Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells.

Resistin-like molecule α stimulates proliferation of mesenchymal stem cells while maintaining their multipotency.

Resistin-like molecule α (RELMα) is highly upregulated in the lungs of mice subjected to hypoxia. It is secreted from pulmonary epithelium and causes potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. By using bone marrow transplantation in mice, we previously showed that RELMα is able to increase the number of bone marrow-derived cells in lung tissue, especially in the remodeling pulmonary vasculature. The current study investigated the effect of RELMα on progenitor stem cell content in mouse lung. Hypoxia, while stimulating RELMα expression, caused an increase in the number of Sca1(+)/CD45(-) progenitor cells in lungs of wild-type mice, but not in lungs of RELMα knockout mice. An in vitro study with cultured mesenchymal stem cells (MSCs) showed that RELMα induced a robust proliferative response that was dependent on Phosphatidylinositol 3-kinase/Akt and Erk activation. RELMα treatment of MSCs caused upregulation of a large number of genes involved in cell cycle, mitosis, organelle, and cytoskeleton biogenesis, and DNA metabolism. MSCs cultured in RELMα-supplemented media were able to maintain their differentiation potential into adipogenic, osteogenic, or mesenchymal phenotypes, although adipogenic differentiation was partially inhibited. These results demonstrate that RELMα may be involved in stem cell proliferation in the lung, without affecting differentiation potential.

Choosing the right antibody for resistin-like molecule (RELM/FIZZ) family members.

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMß/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMß). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.

Role of Smad2/3 and p38 MAP kinase in TGF-ß1-induced epithelial-mesenchymal transition of pulmonary epithelial cells.

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-ß1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-ß1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-ß1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-ß1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-ß1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-ß1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.

Toll Like Receptors Signaling Pathways as a Target for Therapeutic Interventions.

This review summarizes the key role of Toll-Like Receptor (TLRs) molecules for igniting the immune system. Activated by a broad spectrum of pathogens, cytokines or other specific molecules, TLRs trigger innate immune responses. Published data demonstrate that the targeting and suppression of TLRs and TLR-related proteins with particular inhibitors may provide pivotal treatments for patients with cancer, asthma, sepsis, Crohn's disease and thrombosis. Many drugs that target cytokines act in the late phases of the activated pathways, after the final peptides, proteins or glycoproteins are formed in the cell environment. TLR activity occurs in the early activation of cellular pathways; consequently inhibiting them might be most beneficial in the treatment of human diseases.

Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) recruits bone marrow-derived cells to the murine pulmonary vasculature.

BACKGROUND: Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)(+) transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+) BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+) cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+) and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner. CONCLUSIONS/SIGNIFICANCE: These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.

Long-term dynamics of fecal corticosterone in male great gerbils (Rhombomys opimus Licht.): effects of environment and social demography.

We examined the relationship among seasonal characteristics of climate, food, and population demography (social structure) and fecal corticosterone (CORT) concentrations over 6 yr in adult males of an arid-adapted species, the great gerbil (Rhombomys opimus Licht., Gerbillidae, Rodentia), as a measure of chronic stress in high, low, and recovering population densities. Results showed yearly differences in the seasonal means of CORT, with the highest concentrations in the year of the highest population density. Analysis of year-specific relationships revealed a positive correlation between mean CORT and total precipitation in January and February and a negative correlation with precipitation in March. In the beginning of spring, when gerbils were in maximum reproductive effort, CORT correlated positively with the saturation of burrow systems and with the number of adult females with an adult male. A linear stepwise regression of CORT in individual males in spring seasons of all 6 yr combined after removal of year effects revealed that CORT depended positively on the number of females associated with a single male but negatively on the abundance of annual herbs. Disappearance of adult males was not related to CORT in most cases. We found no correlation between overall mortality from season to season and mean CORT in either spring (March-May) or fall. In fact, we found a highly negative correlation between mean CORT and the proportion of disappeared males at the beginning of spring. Only at the high population density when cases of probable catastrophic mortality of all adults in the group were excluded was CORT of individual males related positively to their disappearance during the summer drought. Our results suggest that desert rodents with irregular population fluctuations are more sensitive to suppression by external factors than by density-dependent mortality mediated by stress. The favorable feeding and climatic conditions may have compensated for density-dependent increases of CORT and the negative effects it might have had on survival.

Protective effect of purinergic agonist ATPgammaS against acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure associated with high morbidity and mortality. Although ALI/ARDS pathogenesis is only partly understood, pulmonary endothelium plays a major role by regulating lung fluid balance and pulmonary edema formation. Consequently, endothelium-targeted therapies may have beneficial effects in ALI/ARDS. Recently, attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface P2Y receptors. We previously described ATP-induced endothelial cell (EC) barrier enhancement via a complex cell signaling and hypothesized endothelial purinoreceptors activation to exert anti-inflammatory barrier-protective effects. To test this hypothesis, we used a murine model of ALI induced by intratracheal administration of endotoxin/lipopolysaccharide (LPS) and cultured pulmonary EC. The nonhydrolyzed ATP analog ATPgammaS (50-100 muM final blood concentration) attenuated inflammatory response with decreased accumulation of cells (48%, P < 0.01) and proteins (57%, P < 0.01) in bronchoalveolar lavage and reduced neutrophil infiltration and extravasation of Evans blue albumin dye into lung tissue. In cell culture model, ATPgammaS inhibited junctional permeability induced by LPS. These findings suggest that purinergic receptor stimulation exerts a protective role against ALI by preserving integrity of endothelial cell-cell junctions.

Regulation of vascular endothelial cell barrier function and cytoskeleton structure by protein phosphatases of the PPP family.

Reversible phosphorylation of cytoskeletal and cytoskeleton-associated proteins is a significant element of endothelial barrier function regulation. Therefore, understanding the mechanisms of phosphorylation/dephosphorylation of endothelial cell cytoskeletal proteins is vital to the treatment of severe lung disorders such as high permeability pulmonary edema. In vivo, there is a controlled balance between the activities of protein kinases and phosphatases. Due to various external or internal signals, this balance may be shifted. The actual balances at a given time alter the phosphorylation level of certain proteins with appropriate physiological consequences. The latest information about the structure and regulation of different types of Ser/Thr protein phosphatases participating in the regulation of endothelial cytoskeletal organization and barrier function will be reviewed here.

Potential protein partners for the human TIMAP revealed by bacterial two-hybrid screening.

BacterioMatch Two-Hybrid System (Stratagene) was applied in order to identify potential human TIMAP interaction proteins in the lung. TIMAP highly expressed in endothelial cells and may be involved in endothelial cytoskeletal and barrier regulation. Seven TIMAP interacting partner proteins were identified. Four of identified proteins: cystein and glycine-rich protein 1, eukaryotic translation elongation factor 2, U5 snRNP-specific protein 116 kD, and solute carrier family 3 member 2 are involved in actin cytoskeleton organization, cell adhesion or translation and transcriptional regulation.

Caldesmon is a cytoskeletal target for PKC in endothelium.

We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15-60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise.

Endothelial cell barrier enhancement by ATP is mediated by the small GTPase Rac and cortactin.

ATP is a physiologically relevant agonist released by various sources, including activated platelets, with complex effects mediated via activation of P(2) purinergic receptors. ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, and EC barrier enhancement evoked by ATP has been described. ATP effects on EC barrier function and vascular permeability, however, remain poorly characterized. Although the mechanisms involved are unclear, we previously identified activation of the small GTPase Rac and translocation of cortactin, an actin-binding protein, as key to EC barrier augmentation induced by simvastatin and sphingosine 1-phosphate and therefore examined the role of these molecules in ATP-induced EC barrier enhancement. ATP induced rapid, dose-dependent barrier enhancement in human pulmonary artery EC as measured by transendothelial electrical resistance, with a peak effect appreciable at 25 min (39% increase, 10 microM) and persisting at 2 h. These effects were associated with rearrangement of the EC actin cytoskeleton, early myosin light chain phosphorylation, and spatially defined (cell periphery) translocation of both Rac and cortactin. ATP (10 microM)-treated EC demonstrated a significant increase in Rac activation relative to controls, with a maximal effect (approximately 4-fold increase) at 10 min. Finally, ATP-induced barrier enhancement was markedly attenuated by reductions of either Rac or cortactin (small interfering RNA) relative to controls. Our results suggest for the first time that ATP-mediated barrier protection is associated with cytoskeletal activation and is dependent on both Rac activation and cortactin.

Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement.

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.

The role of caldesmon in the regulation of endothelial cytoskeleton and migration.

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.

Role of CPI-17 in the regulation of endothelial cytoskeleton.

We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99-L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.

Regulated secretion: SNARE density, vesicle fusion and calcium dependence.

SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs.

Social correlates of stress in adult males of the great gerbil, Rhombomys opimus, in years of high and low population densities.

The great gerbil, Rhombomys opimus, is the most social species in the Gerbillinae. The social structure consists of family groups that occupy isolated systems of burrows consisting of one breeding male, from one to seven females, and juveniles. During a year of peak density and one of density decline, we studied the influence of group size, group composition, local density, and distance to the nearest groups on fecal corticosterone and testosterone concentrations in breeding males. We also examined the relationship of hormone concentrations to the survival of males during the summer drought between the spring and the fall. We found that males differed in concentrations of steroid hormones. Concentrations of testosterone were lower whereas those of corticosterone tended to be higher in a year of high population densities compared with higher testosterone and lower corticosterone in a year with a lower density. This finding suggests that stress may be greater in higher densities because of increased social contact. Stepwise regression analysis revealed a positive and significant influence of the number of adult females in a family group on concentrations of fecal corticosterone and testosterone in adult males. Concentrations of corticosterone were also significantly higher in males that disappeared from family groups between the spring and the fall compared with males still alive in family groups in the fall. There was no change in concentrations of testosterone. These results suggest that social interactions within large family groups may be an important source of stress for adult males.