Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1

Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.     

Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded homodimeric mucin-like glycoprotein on leukocytes that interacts with both P- and E-selectin. In this report we describe the structures of the Ser/Thr-linked O-glycans of PSGL-1 synthesized by HL-60 cells metabolically radiolabeled with 3H-sugar precursors. In control studies, the O-glycans on CD43 (leukosialin), a mucin-like glycoprotein also expressed by HL-60 cells, were analyzed and compared to those of PSGL-1. O-Glycans were released from Ser/Thr residues by mild base/borohydride treatment of purified glycoproteins, and glycan structures were determined by a combination of techniques. In contrast to expectations, PSGL-1 is not heavily fucosylated; a majority of the O-glycans are disialylated or neutral forms of the core-2 tetrasaccharide Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAcOH++ +. A minority of the O-glycans are alpha-1,3-fucosylated that occur as two major species containing the sialyl Lewis x antigen; one species is a disialylated, monofucosylated glycan, and the other is a monosialylated, trifucosylated glycan having a polylactosamine backbone. CD43 lacks the fucosylated glycans found on PSGL-1 and is enriched for the nonfucosylated, disialylated core-2 hexasaccharide. These results demonstrate that PSGL-1 contains unique fucosylated O-glycans that are predicted to be critical for high affinity interactions between PSGL-1 and selectins.     

The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum

The P-selectin glycoprotein ligand-1 (PSGL-1) is a high-affinity ligand of P-selectin on myeloid cells and certain subsets of lymphoid cells. We generated the rat monoclonal antibody (MoAb) 2PH1 that recognizes an epitope within the first 19 amino acids at the N-terminus of the processed form of mouse PSGL-1. This antibody blocks attachment of mouse myeloid cells to P-selectin under both static and flow conditions. Intravenous administration of saturating amounts of 2PH1 reduced the number of rolling leukocytes in venules of the acutely exposed mouse cremaster muscle by 79% (+/-5.7%), whereas an anti-P-selectin MoAb reduced it completely. Examining the effect of the MoAb 2PH1 on the recruitment of neutrophils into chemically inflamed mouse peritoneum showed that blocking PSGL-1 inhibited neutrophil accumulation in the peritoneum by 82% (+/-7%) at 2 hours and by 59% (+/-7.9%) at 4 hours after stimulation. A similar effect was seen with the MoAb against P-selectin. Simultaneous administration of both antibodies at the 4-hour time point blocked neutrophil accumulation by 86% (+/-4.2%), arguing for an additional partner molecule for PSGL-1 besides P-selectin. This is the first demonstration of the importance of PSGL-1 in the recruitment of mouse neutrophils into inflamed tissue.     

Comparison of tethering and rolling of eosinophils and neutrophils through selectins and P-selectin glycoprotein ligand-1

We compared the abilities of selectins and the selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), to support tethering and rolling of eosinophils and neutrophils under physiologic flow conditions. Eosinophils and neutrophils accumulated on P-selectin at similar site densities and rolled at similar velocities, but fewer eosinophils than neutrophils accumulated at any P-selectin density. Compared with neutrophils, few eosinophils accumulated on E-selectin except at high densities, and those cells that did accumulate rolled faster than neutrophils. Examination of the mechanisms for accumulation revealed that eosinophils and neutrophils formed similar numbers of primary tethers to P-selectin, whereas eosinophils formed fewer primary tethers to E-selectin than did neutrophils. Compared with neutrophils, adherent eosinophils also supported fewer leukocyte-leukocyte interactions, resulting in diminished secondary tethers to either P- or E-selectin. Studies with mAbs to L-selectin and PSGL-1 demonstrated that neither cell type used L-selectin to form primary tethers to P- or E-selectin. Both eosinophils and neutrophils used the NH2 terminus of PSGL-1 to form primary tethers to P-selectin, but not to E-selectin. Both cell types used L-selectin and PSGL-1 to promote leukocyte-leukocyte interactions and secondary tethers to P- or E-selectin. However, eosinophils developed significantly fewer secondary interactions, probably because they express less L-selectin than do neutrophils.     

A novel P-selectin glycoprotein ligand-1 monoclonal antibody recognizes an epitope within the tyrosine sulfate motif of human PSGL-1 and blocks recognition of both P- and L-selectin

Interactions between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest "rolling" of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSGL-1 has been shown to be the primary mediator of interactions between neutrophils and P-selectin, but studies on the ability of PSGL-1 to mediate interactions between P-selectin and other subsets of leukocytes have yielded variable and conflicting results. A novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and the specificity of this MoAb was confirmed by both flow cytometric analysis and Western blotting of cells transfected with human PSGL-1. This newly developed MoAb, KPL1, inhibited interactions between P-selectin expressing COS cells and either HL60 cells, neutrophils, or lymphocytes. Furthermore, KPL1 completely inhibited interactions between P-selectin and either purified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophils with E-selectin. In addition, KPL1 blocked interactions between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped to a site within a consensus tyrosine sulfation motif of PSGL-1, previously shown to be essential for interaction with P-selectin and now shown to be essential for interaction with L-selectin, and to be distinct from the epitope identified by the PL1 function blocking anti-PSGL-1 MoAb. Two-color flow cytometry of normal leukocytes showed that while natural killer (NK) cells (CD16(+)), monocytes, CD4 and CD8 T cells, and alpha/beta and gamma/delta T cells were uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immunohistologically in germinal centers, which had no detectable KPL1 staining, whereas T-cell areas (interfollicular region) were positive for KPL1. Interestingly, plasma cells in situ and interleukin-6-dependent myeloma cell lines were KPL1(+). Thus, PSGL-1 is expressed on essentially all blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affect the ability of B cells to interact with P- and L-selectin.     

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.     

Effects of Carolina rinse and adenosine rinse on microvascular perfusion and intrahepatic leukocyte-endothelium interaction after liver transplantation in the rat

Flushing hepatic grafts immediately before revascularization with a specially designed rinse solution such as "Carolina rinse" has been reported to improve survival after liver transplantation in the rat. This study investigated the influence of Carolina rinse and adenosine rinse on early graft function, microcirculation, and leukocyte (WBC)-endothelial cell interaction of arterialized syngeneic orthotopic liver transplants in Lewis rats. Livers were preserved for 24 hr in University of Wisconsin solution and flushed immediately before reperfusion with either Ringer's lactate (group A: n = 7), Ringer's lactate + 0.2 mmol/liter adenosine (group B: n = 6), or Carolina rinse (group C: n = 7). Microvascular perfusion and WBC accumulation were assessed by intravital fluorescence microscopy. In group C, acinar perfusion was significantly improved, accompanied by a lower percentage of nonperfused sinusoids 1 hr after reperfusion (mean +/- SEM: 26 +/- 2% [group A], 21 +/- 2% [B], 11 +/- 1% [C], P < 0.01 for C vs. A or B). In addition, Carolina rinse and, to a lesser extent, adenosine rinse reduced the number of WBC sticking in sinusoids and postsinusoidal venules. Better graft function in group C was indicated by increased bile flow during the observation period of 90 min after reperfusion (0.5 +/- 0.3 ml/100 g liver [group A], 1.5 +/- 0.7 [B], 3.7 +/- 0.6 [C], P < 0.01 for C vs. A or B). We conclude that Carolina rinse is able to improve early excretory hepatocellular function, microvascular perfusion, and intrahepatic WBC accumulation after prolonged cold ischemia and reperfusion, but adenosine is unlikely to be the key component of this rinse solution.     

Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.     

Hyporeactivity of rat diaphragmatic arterioles after exposure to hypoxia in vivo. Role of the endothelium

The effect of prior in vivo hypoxia on the in vitro responses to changes in transmural pressure, alpha-adrenoceptor activation, and depolarization with KCl were evaluated in first-order diaphragmatic arterioles. Rats (n = 14 per group) were exposed to normoxia (controls) or to hypoxia (inspired O2 concentration = 10%) for 12 or 48 h. The arteriolar pressure-diameter relationships were recorded over a pressure range from 10 to 200 mm Hg. In separate groups of arterioles (n = 12 per group), the diaphragmatic arteriolar responses to phenylephrine (10(-8) to 10(-5 M) or KCl (10 to 100 mM) were determined after exposure to either room air or hypoxia for 48 h. In half of the arterioles studied, the endothelium was removed. After 12 h of hypoxia, the pressure-diameter relationship was normal in endothelialized arterioles but was shifted upward in de-endothelialized vessels (p < 0.05). After 48 h of hypoxia, the constrictor response to increasing transmural pressure was severely suppressed in all arterioles. The intraluminal diameters during activation with phenylephrine and KCl were larger in arterioles from rats exposed to hypoxia (103 +/- 8 and 81 +/- 7 microns, respectively) than in control arterioles (41 +/- 5 and 54 +/- 6 microns, respectively; p < 0.05 for differences). During maximum phenylephrine- and KCl-induced constriction in de-endothelialized arterioles, diameters averaged 125 +/- 8 and 105 +/- 8 microns, respectively, for arterioles from hypoxic rats and 32 +/- 6 and 40 +/- 5 microns, respectively, for arterioles from control vessels. Exposure to hypoxia results in impairment of diaphragmatic arteriolar smooth muscle reactivity and reversal of the normal inhibitory influence of the endothelium on diaphragmatic arteriolar tone.     

Effect of serotonin on unidirectional ion fluxes in rat intestine in vivo

AIMS: A stimulating intestinal secretory effect is described in vitro and an inhibition with selective inhibitors of the different receptors of serotonin (5-HT), in vivo. But a direct effect, in vivo, in fully vascularized and innervated intestine has not yet been clearly evidenced. We studied the effect of 5-HT in anesthetized rats with ligated loops. This work, performed at 4 intestinal levels, allowed a comparison with the effects of a known stimulant of intestinal secretion, VIP, and a specific inhibitor of Na/H exchange, dimethylamiloride (DMA). RESULTS: 5-HT induced an inhibition of epithelial Na influx in agreement with the inhibition of Na/H exchanger, an inhibition of the influx of Cl, partially passive absorption following Na by paracellular route. A decrease of Na and Cl efflux was induced by 5-HT in duodenum, jejunum and ileum while in colon, a stimulation was obtained by intraluminal but not intravenous route. CONCLUSION: Even though 5-HT induced a liquid accumulation in all intestinal segments, the effect differed according to the intestinal level, either inhibition of absorption in the small intestine, or stimulation of secretion in the colon. The comparison of the effect of 5-HT with that of DMA shows that the inhibition of absorption is not only due to Na/H exchanger inhibition.     

Effects of a metalloproteinase that truncates P-selectin glycoprotein ligand on neutrophil-induced cardiac dysfunction in ischemia/reperfusion

This study was designed to test the effects of polymorphonuclear leukocytes (PMNs) in the presence and absence of a P-selectin blocker, mocarhagin, in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Control rat hearts not subjected to ischemia were perfused without blood cells for 80 min. Additional control rat hearts were perfused with 100 x 10(6) PMNs in the presence and absence of 0.2 microgram/ml mocarhagin over a 5-min perfusion followed by a 45-min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dt max) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 min of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of PMNs. I/R hearts perfused with PMNs exhibited decreases of 50-60% in all measurements of cardiac function (P < 0.001). These PMN perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selection expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were attenuated markedly by the metalloproteinase, mocarhagin, which inhibits P-selectin-mediated cell adhesion by cleaving its high-affinity receptor, PSGL-1, present on neutrophils. These results provide evidence that neutrophils provoke post-reperfusion cardiac dysfunction, and that this may be largely due to P-selectin-induced adherence of neutrophils to the endothelium.     

Role of platelet-activating factor and prostanoids in hemodynamic changes in rat experimental endotoxic shock

BACKGROUND: Gliadin amino acid sequence(s) responsible for toxicity in susceptible individuals have not been fully elucidated. Previous in vitro studies have suggested the presence of active sequences in the NH(2)-terminal part of the A-gliadin molecule. In this paper the in vitro activity of A-gliadin synthetic peptides 31-55, 31-43, and 44-55 has been investigated. METHODS: Organ culture of jejunal mucosa from untreated and treated coeliac patients was used. In the first system enterocyte height was used as a measure of peptide toxicity; in the second system evidence of activated mucosal cell-mediated immune response was sought. RESULTS: Peptides 31-55 and 31-43 were active on untreated coeliac mucosa at a concentration of 0.5 mg/ml and peptide 44-55 only at a concentration of 3 mg/ml. In in vitro-cultured treated coeliac mucosa peptides 31-55 and 31-43 at 1 mg/ml and peptide 44-55 at 3 mg/ml were able to induce enhanced epithelial expression of HLA-DR and 4F2 molecules and the appearance of CD25 positive cells. CONCLUSIONS: Our results suggest that 31-43 and 44-55 A-gliadin peptides are both active, even if to different extents. In vitro systems remain essential tools to screen material to be subsequently tested in vivo.     

Effect of music therapy on mood and social interaction among individuals with acute traumatic brain injury and stroke.

Objective: To investigate the efficacy of music therapy techniques as an aid in improving mood and social interaction after traumatic brain injury or stroke. Design: Eighteen individuals with traumatic brain injury or stroke were assigned either standard rehabilitation alone or standard rehabilitation along with music therapy (3 treatments per week for up to 10 treatments). Measures: Pretreatment and posttreatment assessments of participant self-rating of mood, family ratings of mood and social interaction, and therapist rating of mood and participation in therapy. Results: There was a significant improvement in family members' assessment of participants' social interaction in the music therapy group relative to the control group. The staff rated participants in the music therapy group as more actively involved and cooperative in therapy than those in the control group. There was a trend suggesting that self-ratings and family ratings of mood showed greater improvement in the music group than in the control group. Conclusions: Results lend preliminary support to the efficacy of music therapy as a complementary therapy for social functioning and participation in rehabilitation with a trend toward improvement in mood during acute rehabilitation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of subliminal shock conditioning on recall.

Presented words at chance recognition levels and paired them with either the abrupt initiation or cessation of shock conditioning procedures. 18 male graduate students served as Ss in each of 3 experiments. Exp. I showed that words associated with abrupt initiation of shock conditioning appeared later in free recall than words associated with cessation of shock conditioning. Exp. II replicated the results of Exp. I when S's task during conditioning was irrelevant to recognizing words. Exp. III, using 50% of the stimulus intensity of Exp. I and II, demonstrated residual differentiation in recall between words actually presented and those whose presentation was simulated. Results are interpreted as evidence for subliminal perception. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Role of stigma and set in interpersonal interaction.

This study focuses on 1 condition which seems to influence the role of stigmata in interpersonal relationships. The results indicate that mental illness and the extent to which a person is held responsible for this stigma determine the amount of pain inflicted upon him and how favorably he is evaluated. Responsibility was varied by having a confederate report to the undergraduate Ss either a typical or an atypical and pathogenic childhood. A bad childhood mitigated the harshness of the treatment accorded the mentally ill person, but for the normal individual this experience appears to be as stigmatizing as mental illness itself. The individual perceived as abnormal either because of mental illness or a poor childhood experience is treated in a harsher manner than the normal. He is also described as less adequate in his performance, although there is no objective basis for this, he is less liked, and Ss prefer no further interaction with him. An interesting implication of comparing the evaluation with the behavior displayed is that people can be induced to behave more favorably while retaining all of their dislike and contempt for the stigmatized person. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Release of soluble intercellular adhesion molecule 1 into bile and serum in murine endotoxin shock

Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecules Mac-1 (CD11b/CD18) on neutrophils and its counterreceptor on endothelial cells and hepatocytes, intercellular adhesion molecule 1 (ICAM-1). To investigate a potential release of a soluble form of ICAM-1 (sICAM-1), animals received 100 micrograms/kg Salmonella abortus equi endotoxin alone or in combination with 700 mg/kg galactosamine. In endotoxin-sensitive mice (C3Heb/FeJ), injection of endotoxin did not cause liver injury but induced a time-dependent increase of sICAM-1 in serum (300%) and in bile (615%) without affecting bile flow. In galactosamine/endotoxin-treated animals, which developed liver injury, the increase in both compartments was only 97% and 104%, respectively. In either case, the increase in sICAM-1 concentrations paralleled the enhanced ICAM-1 expression in the liver. The endotoxin-resistant strain (C3H/HeJ) did not show elevated sICAM-1 levels in serum or bile after endotoxin administration. In contrast, the intravenous injection of murine tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) or IL-1 beta (13-23 micrograms/kg) into endotoxin-resistant mice induced a 225% to 364% increase in serum sICAM-1 and a 370% elevation of the biliary efflux of sICAM-1, again independent of changes in bile flow. These data indicate that cytokines are major inducers of sICAM-1 formation during endotoxemia in vivo. The described experimental model can be used to investigate the role of sICAM-1 in the pathophysiology of inflammatory liver disease.     

Effect of sorbin derivatives on cholera toxin-induced intestinal secretion in rat in vivo

The effect of synthetic sorbin derivatives was determined on cholera toxin-stimulated jejunal secretion in anesthetized rats in vivo, using both perfused and ligated loop. An inhibitory effect on water secretion induced by cholera toxin was shown with C-terminal sorbin peptides: C20 (YEPGKSSILQHERPVTKPQA-amide), C10 and Dala 7 heptapeptide-amide of sorbin, given by subcutaneous (SC) or intraduodenal administration. When perfused intravenously, C20-sorbin inhibited the cholera-induced stimulation of net flux of water, Na+ and K+, in the jejunum and at the same time the net flux of water and Cl- in the duodenum, which was not in contact with the toxin. 5-hydroxytryptamine was not significantly changed in plasma or fluid. Prostaglandin E2 release in jejunal as well as duodenal fluid was significantly stimulated by cholera toxin, but was not significantly different from basal value after C20 administration.     

Leukocyte-endothelium interaction during the early stages of hypercholesterolemia in the rabbit: role of P-selectin, ICAM-1, and VCAM-1

The early effects of hypercholesterolemia on leukocyte-endothelium interaction were studied in vivo in the rabbit mesenteric microcirculation. Rabbits fed a 0.5% high-cholesterol (HC) diet showed elevated plasma cholesterol levels during the 1 to 2 weeks of HC feeding (P<0.001 versus control diet-fed rabbits). Intravital microscopy of mesenteric venules revealed that leukocyte rolling had increased 10-fold (P<0.001 versus control-fed group) at the end of the first week of the HC diet, which was sustained after 2 weeks of HC feeding (P<0.001 versus control-fed rabbits). Firm adherence of leukocytes to the endothelium was moderately increased after a 1-week period of hypercholesterolemia (P<0.05) but increased by 12-fold at 2 weeks (P<0.001 versus control diet-fed and P<0.01 versus 1-week HC-fed rabbits). Upregulation of the endothelial cell adhesion molecules P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was observed immunohistochemically on the intestinal microvascular endothelium of HC-fed rabbits. P-selectin was maximally expressed within the first week of the HC diet and remained elevated during the second week of cholesterol feeding (P<0.01 versus control). In contrast, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were moderately upregulated at 1 week but were highly expressed after 2 weeks of the HC diet (P<0.05 and P<0.001 versus control, respectively). Basal release of NO from both mesenteric microvascular and aortic endothelium in cholesterol-fed rabbits was progressively reduced after 1 (P<0.05) and 2 (P<0.01) weeks. Our data suggest that enhanced leukocyte-endothelium interaction occurs in vivo in the rabbit microcirculation during the first 2 weeks of hypercholesterolemia. This phenomenon is associated with impaired basal NO release and progressive endothelial surface expression of endothelial cell adhesion molecules (ie, P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1) in the microvasculature.     

Effect of in vivo hyperoxia on the glutathione system in neonatal rat lung

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.