Structure and function of the selectin ligand PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAca2®3Galß1®4[Fuca1®3]GlcNAcß1®R). In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

Perturbation of EGF-induced MAP kinase activation by TGF-ß1

TGF-ß1 regulates both cellular growth and phenotypic plasticity important for maintaining a growth advantage and increased invasiveness in progressively malignant cells. Recent studies indicate that TGF-ß-1 stimulates the conversion of epitheliod to fibroblastoid phenotype which presumably leads to the inactivation of growth-inhibitory effects by TGF-ß1 (Portella et al. (1998) Cell Growth and Differentiation, 9: 393-404). Therefore, the investigation of TGF-ß1 signaling that leads to altered growth and migration may provide novel targets for the prevention of increased cell growth and invasion. Although much attention has been paid to TGF-ß1 responses in epithelial cells, the above studies suggest that examination of signal transduction pathways in fibroblasts are important as well. Data from our laboratory are consistent with the concept that TGF-ß1 can act as a regulatory switch in density-dependent C3H 10T1/2 fibroblasts capable of either promoting or delaying G1 traverse. The regulation of this switch is proposed to occur prior to pRb phosphorylation, namely prior to activation of cyclin-dependent kinases. The current study is concerned with the evaluation of a key cyclin (cyclin D1) which activates cdk4 and p27KIP1 which in turn inhibit cdk2 in the proliferative responses of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and their modulation by TGF-ß1. Although the molecular events that lead to elevation of cyclin D1 are not completely understood, it appears likely that activation of p42/p44MAPK kinases is involved in its transcriptional regulation. TGF-ß1 delayed EGF- or PDGF-induced cyclin D1 expression and blocked the induction of active p42/p44MAPK. The mechanism by which TGF-ß1 induces a block in p42/p44MAPK activation is being examined and the possibility that TGF-ß1 regulates phosphatase activity is being tested.

Residual ß-cell function and microvascular complications in type 1 diabetic patients

To determine the influence of residual ß-cell function on retinopathy and microalbuminuria we measured basal C-peptide in 50 type 1 diabetic outpatients aged 24.96 ± 7.14 years, with a duration of diabetes of 9.1 ± 6.2 years. Forty-three patients (86%) with low C-peptide (<0.74 ng/ml) had longer duration of diabetes than 7 patients (14%) with high C-peptide (³0.74 ng/ml) (9 (2-34) vs 3 (1-10) years, P = 0.01) and a tendency to high glycated hemoglobin (HBA1) (8.8 (6-17.9) vs 7.7 (6.9-8.7)%, P = 0.08). Nine patients (18%) had microalbuminuria (two out of three overnight urine samples with an albumin excretion rate (AER) ³20 and <200 µg/min) and 13 (26%) had background retinopathy. No association was found between low C-peptide, microalbuminuria and retinopathy and no difference in basal C-peptide was observed between microalbuminuric and normoalbuminuric patients (0.4 ± 0.5 vs 0.19 ± 0.22 ng/ml, P = 0.61) and between patients with or without retinopathy (0.4 ± 0.6 vs 0.2 ± 0.3 ng/ml, P = 0.43). Multiple regression analysis showed that duration of diabetes (r = 0.30, r2 = 0.09, P = 0.031) followed by HBA1 (r = 0.41, r2 = 0.17, P = 0.01) influenced basal C-peptide, and this duration of diabetes was the only variable affecting AER (r = 0.40, r2 = 0.16, P = 0.004). In our sample of type 1 diabetic patients residual ß-cell function was not associated with microalbuminuria or retinopathy.

Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.

Blockade of NK-1 receptors in the lateral commissural nucleus tractus solitarii of awake rats had no effect on the cardiovascular responses to chemoreflex activation

The neurotransmission of the chemoreflex in the nucleus tractus solitarii (NTS), particularly of the sympatho-excitatory component, is not completely understood. There is evidence that substance P may play a role in the neurotransmission of the chemoreflex in the NTS. Microinjection of substance P (50 pmol/50 nl, N = 12, and 5 nmol/50 nl, N = 8) into the commissural NTS of unanesthetized rats produced a significant increase in mean arterial pressure (101 ± 1 vs 108 ± 2 and 107 ± 3 vs 115 ± 4 mmHg, respectively) and no significant changes in heart rate (328 ± 11 vs 347 ± 15 and 332 ± 7 vs 349 ± 13 bpm, respectively) 2 min after microinjection. Previous treatment with WIN, an NK-1 receptor antagonist (2.5 nmol/50 nl), microinjected into the NTS of a specific group of rats, blocked the pressor (11 ± 5 vs 1 ± 2 mmHg) and tachycardic (31 ± 6 vs 4 ± 3 bpm) responses to substance P (50 pmol/50 nl, N = 5) observed 10 min after microinjection. Bilateral microinjection of WIN into the lateral commissural NTS (N = 8) had no significant effect on the pressor (50 ± 4 vs 42 ± 6 mmHg) or bradycardic (-230 ± 16 vs -220 ± 36 bpm) responses to chemoreflex activation with potassium cyanide (iv). These data indicate that the activation of NK-1 receptors by substance P in the NTS produces an increase in baseline mean arterial pressure and heart rate. However, the data obtained with WIN suggest that substance P and NK-1 receptors do not play a major role in the neurotransmission of the chemoreflex in the lateral commissural NTS.

Insights into the physiological function of cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction.

Angiotensin-(1-7) improves the post-ischemic function in isolated perfused rat hearts

We evaluated the effects of angiotensin-(1-7) (Ang-(1-7)) on post-ischemic function in isolated hearts from adult male Wistar rats perfused according to the Langendorff technique. Local ischemia was induced by coronary ligation for 15 min. After ischemia, hearts were reperfused for 30 min. Addition of angiotensin II (Ang II) (0.20 nM, N = 10) or Ang-(1-7) (0.22 nM, N = 10) to the Krebs-Ringer perfusion solution (KRS) before the occlusion did not modify diastolic or systolic tension, heart rate or coronary flow (basal values for Ang-(1-7)-treated hearts: 0.72 ± 0.08 g, 10.50 ± 0.66 g, 216 ± 9 bpm, 5.78 ± 0.60 ml/min, respectively). During the period of occlusion, the coronary flow, heart rate and systolic tension decreased (values for Ang-(1-7)-treated hearts: 2.83 ± 0.24 ml/min, 186 ± 7 bpm, 6.95 ± 0.45 g, respectively). During reperfusion a further decrease in systolic tension was observed in control (4.95 ± 0.60 g) and Ang II-treated hearts (4.35 ± 0.62 g). However, in isolated hearts perfused with KRS containing Ang-(1-7) the further reduction of systolic tension during the reperfusion period was prevented (7.37 ± 0.68 g). The effect of Ang-(1-7) on the systolic tension was blocked by the selective Ang-(1-7) antagonist A-779 (2 nM, N = 9), by the bradykinin B2 antagonist HOE 140 (100 nM, N = 10), and by indomethacin pretreatment (5 mg/kg, ip, N = 8). Pretreatment with L-NAME (30 mg/kg, ip, N = 8) did not change the effect of Ang-(1-7) on systolic tension (6.85 ± 0.61 g). These results show that Ang-(1-7) at low concentration (0.22 nM) improves myocardial function (systolic tension) in ischemia/reperfusion through a receptor-mediated mechanism involving release of bradykinin and prostaglandins.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3% positive cells), alpha3 (93.3 ± 7.0%), alpha5 (50.4 ± 12.0%) and alpha6 (34.1 ± 4.9%) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0%) and fibronectin (40.0 ± 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4% vs DMSO: 70.7 ± 2.5%) while simultaneously reducing alpha5 (24.2 ± 19%) and alpha6 (14.3 ± 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 ± 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 ± 2.4 and 6.7 ± 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 ± 0.97 and 7.5 ± 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 µM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 µM L-NOARG from 73 ± 5.1 to 19 ± 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 ± 5.85% (from 162 ± 10 to 73 ± 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 ± 2.7 vs 91.2 ± 0.9%, P < 0.02; 35.96 ± 11.64 vs 22.19 ± 9.65%, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 µM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

Imbalance of naive and memory T lymphocytes with sustained high cellular activation during the first year of life from uninfected children born to HIV-1-infected mothers on HAART

The immune consequences of in utero HIV exposure to uninfected children whose mothers were submitted to highly active antiretroviral therapy (HAART) during gestation are not well defined. We evaluated 45 HIV-exposed uninfected (ENI) neonates and 45 healthy unexposed control (CT) neonates. All HIV-infected mothers received HAART during pregnancy, and the viral load at delivery was <50 copies/mL for 56.8%. Twenty-three ENI neonates were further evaluated after 12 months and compared to 23 unexposed healthy age-matched infants. Immunophenotyping was performed by flow cytometry in cord and peripheral blood. Cord blood lymphocyte numbers did not differ between groups. However, ENI neonates had a lower percentage of naive T cells than CT neonates (CD4+, 76.6 vs 83.1%, P < 0.001; CD8+, 70.9 vs 79.6%, P = 0.003) and higher percentages of central memory T cells than CT neonates (CD4+, 13.9 vs 8.7%, P < 0.001; CD8+, 8.6 vs 4.8%, P = 0.001). CD38 mean fluorescence intensity of T cells was higher in ENI neonates (CD4+, 62.2 vs 52.1, P = 0.007; CD8+, 47.7 vs 35.3, P < 0.001). At 12 months, ENI infants still had higher mean fluorescence intensity of CD38 on T cells (CD4+, 34.2 vs 23.3, P < 0.001; CD8+, 26.8 vs 19.4, P = 0.035). Despite effective maternal virologic control at delivery, HIV-exposed uninfected children were born with lower levels of naive T cells. Immune activation was present at birth and remained until at least 12 months of age, suggesting that in utero exposure to HIV causes subtle immune abnormalities.

Dermatan sulfate reduces monocyte chemoattractant protein 1 and TGF-β production, as well as macrophage recruitment and myofibroblast accumulation in mice with unilateral ureteral obstruction

Selectins play an essential role in most inflammatory reactions, mediating the initial leukocyte-rolling event on activated endothelium. Heparin and dermatan sulfate (DS) bind and block P- and L-selectin function in vitro. Recently, we reported that subcutaneous administration of DS inhibits colon inflammation in rats by reducing macrophage and T-cell recruitment and macrophage activation. In the present study, we examined the effect of porcine intestinal mucosa DS on renal inflammation and fibrosis in mice after unilateral ureteral obstruction (UUO). Twenty-four adult male Swiss mice weighing 20-25 g were divided into 4 groups: group C (N = 6) was not subjected to any surgical manipulation; group SH (N = 6) was subjected to surgical manipulation but without ureter ligation; group UUO (N = 6) was subjected to unilateral ureteral obstruction and received no treatment; group UUO plus DS (N = 6) was subjected to UUO and received DS (4 mg/kg) subcutaneously daily for 14 days. An immunoblot study was also performed for TGF-β. Collagen (stained area ~3700 µm²), MCP-1 (stained area ~1700 µm²), TGF-β (stained area ~13% of total area), macrophage (number of cells ~40), and myofibroblast (stained area ~1900 µm²) levels were significantly (P < 0.05) higher in the UUO group compared to control. DS treatment significantly (P < 0.05) reduced the content of collagen (stained area ~700 µm²), MCP-1 (stained area ~160 µm²) and TGF-β (stained area ~5% of total area), in addition to myofibroblast (stained area ~190 µm²) and macrophage (number of cells ~32) accumulation in the obstructed kidney. Overall, these results indicate that DS attenuates kidney inflammation by reducing macrophage recruitment, myofibroblast population and fibrosis in mice submitted to UUO.

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.