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Lymphocyte integrins mediate cell arrest on endothelium during immune surveillance after activation by chemokine-stimulated inside-out signals. Here we show that a Vav1-talin complex in T cells is a key target for chemokine-triggered inside-out signaling leading to integrin alpha4beta1 activation. Thus, Vav1 dissociation from talin was required to generate high-affinity alpha4beta1 conformations. Assembly of the Vav1-talin complex required PtdIns(4,5)P(2), which was provided by talin-bound phosphatidylinositol phosphate kinase Igamma. Chemokine-promoted Vav1 dissociation from talin followed an initial increase in talin binding to alpha4beta1. This process was dependent on ZAP-70, which binds to and phosphorylates Vav1 in the complex, leading to further alpha4beta1 activation and cell adhesion strengthening. Moreover, Vav1-talin dissociation was needed for Rac1 activation, thus indicating that alpha4beta1 and Rac1 activation can be coupled by chemokine-stimulated ZAP-70 function. Our data suggest that Vav1 might function as a repressive adaptor of talin that must dissociate from alpha4beta1-talin complexes for efficient integrin activation., (Copyright 2009 Elsevier Inc. All rights reserved.)

Tumour cell dissemination through corporal fluids (blood, lymph and body cavity fluids) is a distinctive feature of the metastatic process. Tumour cell transition from fluid to adhesive conditions involves an early polarization event and major rearrangements of the submembrane cytoskeleton that remain poorly understood. As regulation of cortical actin-membrane binding might be important in this process, we investigated the role of ezrin and moesin, which are key crosslinking proteins of the ERM (ezrin, radixin, moesin) family. We used short interfering RNA (siRNA) to show that moesin is crucial for invasion by melanoma cells in 3D matrices and in early lung colonization. Using live imaging, we show that following initial adhesion to the endothelium or 3D matrices, moesin is redistributed away from the region of adhesion, thereby generating a polarized cortex: a stable cortical actin dome enriched in moesin and an invasive membrane domain full of blebs. Using Lifeact-GFP, a 17-amino-acid peptide that binds F-actin, we show the initial symmetry breaking of cortical actin cytoskeleton during early attachment of round cells. We also demonstrated that ezrin and moesin are differentially distributed during initial invasion of 3D matrices, and, specifically, that moesin controls adhesion-dependent activation of Rho and subsequent myosin II contractility. Our results reveal that polarized moesin plays a role in orienting Rho activation, myosin II contractility, and cortical actin stability, which is crucial for driving directional vertical migration instead of superficial spreading on the fluid-to-solid tissue interface. We propose that this mechanism of cortical polarization could sustain extravasation of fluid-borne tumour cells during the process of metastasis.

Aim: To evaluate the effect of gravitational valves on over-drainage in hydrocephalus in adults., Patients and Methods: We performed a retrospective study of the shunt systems placed in patients over the age of 18 years between 1998 and 2006. Patients were divided into two groups: non-GV group (without gravitational valve) and GV group (with a gravitational valve, Aesculap-Miethke 5/35). The complications that occurred during the first year following the placement of the shunt system were recorded., Results: Of a total of 137 patients, 91 were from the non-GV group and 46 belonged to the GV group. Mean age: non-GV group, 62.1 years; and GV group, 64.2 years, without any significant differences. In 80 patients the aetiology was chronic adult hydrocephalus, 19 were due to expansive processes, 15 due to vascular causes, eight pseudo tumours, six post-traumatic injuries and nine were due to other causes. In the non-GV group, 9.89% presented over-drainage, whereas there were no cases in the GV group; the difference was statistically significant (p = 0.029). In the rest of the complications there were no significant differences between the two groups. The total complications in the non-GV group were 25.27% and in the GV group, 6.52%, and there were significant differences (p = 0.01), although, above all, at the expense of over-drainage, because if this complication was excluded, then the differences were no longer significant (p = 0.175)., Conclusions: In our series, the use of gravitational valves in the prevention of over-drainage in adult hydrocephalus proved to be more effective than employing valves without the gravitational device.

Molecular requirements and determinants for efficient binding to CCR5 were interpreted by computational techniques based on comparative receptor structure modeling, advanced 3D-QSAR, docking, and shape-based virtual screening of commercially available entry blockers. Results of this study may be valuable for predicting new HIV entry-blocking leads.Acquired immune deficiency syndrome (AIDS) is responsible for more than 31 million deaths, and many more people are affected by this disease worldwide. Novel ligands that are capable of blocking virus-cell fusion are emerging as promising candidate molecules against HIV-1 infection because they have the promise to overcome the major drawbacks of classical highly active antiretroviral (HAART) drugs. However, structure-based design continues to be hampered owing to the paucity of experimentally determined 3D information about HIV-1 cell-surface co-receptors. Using computational techniques based on comparative receptor structure modeling, advanced 3D-QSAR, and protein-ligand docking, we present recent results that define updated molecular requirements and determinants for efficient binding of small-molecule ligands to CCR5, a principal biological target for HIV entry blockers. These results are compared with shape- and property-based virtual screening results for commercially available entry blockers, and will be valuable for predicting new HIV entry-blocking leads.

Melanoma cells express the chemokine receptor CXCR4 that confers high invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial stages of the disease show reduction or loss of E-cadherin expression, but recovery of its expression is frequently found at advanced phases. We overexpressed E-cadherin in the highly invasive BRO lung metastatic cell melanoma cell line to investigate whether it could influence CXCL12-promoted cell invasion. Overexpression of E-cadherin led to defective invasion of melanoma cells across Matrigel and type I collagen in response to CXCL12. A decrease in individual cell migration directionality toward the chemokine and reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent inhibition of RhoA activation was responsible for the impairment in chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore, we show that p190RhoGAP and p120ctn associated predominantly on the plasma membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association. These results suggest that melanoma cells at advanced stages of the disease could have reduced metastatic potency in response to chemotactic stimuli compared with cells lacking E-cadherin, and the results indicate that p190RhoGAP is a central molecule controlling melanoma cell invasion.

A new interaction fingerprint (IF) called APIF (atom-pairs-based interaction fingerprint) has been developed for postprocessing protein-ligand docking results. Unlike other existing fingerprints which employ absolute locations of individual interactions, APIF considers the relative positions of pairs of interacting atoms. Docking-based virtual screening was performed with GOLD using the crystal structures of trypsin, rhinovirus, HIV protease, carboxypeptidase, and estrogen receptor-alpha as targets. A score derived from the similarity of the bit strings for each docking solution to that of a known reference binding mode was obtained. Comparisons between APIF, GoldScore function, and standard interaction fingerprint (CHIF) scores were performed using enrichment plots. Superior recovery rates were observed in the IF score cases. Comparable results were achieved by using either of the two interaction fingerprints, substantially improving GoldScore function enrichment factors. Binding mode analyses were also carried out in order to study the best method for selecting conformations with a binding mode similar to that of the reference crystallized complex. These showed that the first conformations retrieved by interaction fingerprint scores had a more similar binding mode to the reference complex than those retrieved by the GoldScore function.

The process of HIV entry begins with the binding of the viral envelope glycoprotein gp120 to both the CD4 receptor and one of CXCR4 or CCR5 chemokine coreceptors. There is currently considerable interest in developing novel ligands which can attach to these coreceptors and hence block virus-cell fusion. This article compares the application of structure-based (docking) and ligand-based (QSAR analyses, pharmacophore modeling, and shape matching) virtual screening tools to find new potential HIV entry inhibitors for the CXCR4 receptor. The comparison is based on retrospective virtual screening of a library containing different known CXCR4 inhibitors from the literature, a smaller set of active CXCR4 inhibitors selected from a large combinatorial virtual library and synthesized by us, and some druglike presumed inactive molecules as the reference set. The enrichment factors and diversity of the retrieved molecular scaffolds in the virtual hit lists was determined. Once the different virtual screening approaches had been validated and the best parameters had been selected, prospective virtual screening of our virtual library was applied to identify new anti-HIV compounds using the same protocol as in the retrospective virtual screening analysis. The compounds selected using these computational tools were subsequently synthesized and assayed and showed activity values ranging from 4 to 0.022 microg/mL.

Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma.

A protocol for the synthesis of N-substituted 2-hydro-4-amino-pyrido[2,3-d]pyrimidin-7(8H)-ones (11) is described. Thus, the formylation of a 2-aminopyridone 12 in 85% formic acid/Ac(2)O, proceeding via in situ cyclization to the intermediate formamide 13, affords the corresponding 2-hydro-4-oxo-pyridopyrimidine 14, which is converted to a 4-chloro-pyridopyrimidine 15 upon treatment with POCl(3). The subsequent transformation to the title compounds is carried by treatment with the corresponding amine in MeOH under microwave irradiation conditions.

HIV entry inhibitors have emerged as a new generation of antiretroviral drugs that block viral fusion with the CXCR4 and CCR5 membrane coreceptors. Several small molecule antagonists for these coreceptors have been developed, some of which are currently in clinical trials. However, because no crystal structures for the coreceptor proteins are available, the binding modes of the known inhibitors within the coreceptor extracellular pockets need to be analyzed by means of site-directed mutagenesis and computational experiments. Previous studies have indicated that there is more than one binding site within the CCR5 extracellular pocket. This article investigates and develops this hypothesis using a novel spherical harmonic-based consensus shape clustering approach. The consensus shape approach is evaluated using retrospective virtual screening of CXCR4 and CCR5 inhibitors. Multiple combinations of CCR5 ligands in multiple trial superpositions are constructed to find consensus queries that give high virtual screening enrichments. Receiver-operator-characteristic performance analyses for both CXCR4 and CCR5 inhibitors show that the new consensus shape matching approach gives better virtual screening enrichments than existing shape matching and docking virtual screening techniques. The results obtained also provide strong evidence to support the notion that there are three main binding sites within the CCR5 extracellular cavity.

The GTPase RhoA is a downstream target of heterotrimeric G(13) proteins and plays key roles in cell migration and invasion. Here, we show that expression in human melanoma cells of a constitutively active, GTPase-deficient Galpha(13) form (G(alpha)(13)QL) or lysophosphatidylcholine (LPC)-promoted signaling through G(alpha)(13)-coupled receptors led to a blockade of chemokine-stimulated RhoA activation and cell invasion that was rescued by active RhoA. Melanoma cells expressing G(alpha)(13)QL or cells stimulated with LPC displayed an increase in p190RhoGAP activation, and defects in RhoA activation and invasion were recovered by knocking down p190RhoGAP expression, thus identifying this GTPase-activating protein (GAP) protein as a downstream G(alpha)(13) target that is responsible for these inhibitory responses. In addition, defective stress fiber assembly and reduced migration speed underlay inefficient invasion of G(alpha)(13)QL melanoma cells. Importantly, G(alpha)(13)QL expression in melanoma cells led to impairment in lung metastasis associated with prolonged survival in SCID mice. The data indicate that G(alpha)(13)-dependent downstream effects on RhoA activation and invasion tightly depend on cell type-specific GAP activities and that G(alpha)(13)-p190RhoGAP signaling might represent a potential target for intervention in melanoma metastasis.

HIV cell fusion and entry have been validated as targets for therapeutic intervention against infection. Bicyclams were the first low-molecular-weight compounds to show specific interaction with CXCR4. The most potent bicyclam was AMD3100, in which the two cyclam moieties are tethered by a 1,4-phenylenebis(methylene) bridge. It was withdrawn from clinical trials owing to its lack of oral bioavailability and cardiotoxicity. We have designed a combinatorial library of non-cyclam polynitrogenated compounds by preserving the main features of AMD3100. At least two nitrogen atoms on each side of the p-phenylene moiety, one in the benzylic position and the other(s) in the heterocyclic system were maintained, and the distances between them were similar to the nitrogen atom distances in cyclam. A selection of diverse compounds from this library were prepared, and their in vitro activity was tested in cell cultures against HIV strains. This led to the identification of novel potent CXCR4 coreceptor inhibitors without cytotoxicity at the tested concentrations.

HIV infection is initiated by fusion of the virus with the target cell through binding of the viral gp120 protein with the CD4 cell surface receptor protein and the CXCR4 or CCR5 co-receptors. There is currently considerable interest in developing novel ligands that can modulate the conformations of these co-receptors and, hence, ultimately block virus-cell fusion. This article describes a detailed comparison of the performance of receptor-based and ligand-based virtual screening approaches to find CXCR4 and CCR5 antagonists that could potentially serve as HIV entry inhibitors. Because no crystal structures for these proteins are available, homology models of CXCR4 and CCR5 have been built, using bovine rhodopsin as the template. For ligand-based virtual screening, several shape-based and property-based molecular comparison approaches have been compared, using high-affinity ligands as query molecules. These methods were compared by virtually screening a library assembled by us, consisting of 602 known CXCR4 and CCR5 inhibitors and some 4700 similar presumed inactive molecules. For each receptor, the library was queried using known binders, and the enrichment factors and diversity of the resulting virtual hit lists were analyzed. Overall, ligand-based shape-matching searches yielded higher enrichments than receptor-based docking, especially for CXCR4. The results obtained for CCR5 suggest the possibility that different active scaffolds bind in different ways within the CCR5 pocket.

Background: Sevelamer hydrochloride is a phosphate binder widely employed in hemodialysis patients. Until now, information about its efficacy and safety in peritoneal dialysis patients has been scarce., Patients and Methods: In September 2005 a cross-sectional study of demographic, biochemical, and therapeutic data of patients from 10 peritoneal dialysis units in Catalonia and the Balearic Islands, Spain, was conducted., Results: We analyzed data from 228 patients. At the time of the study, 128 patients (56%) were receiving sevelamer. Patients receiving sevelamer were younger (p < 0.01), showed a longer period of time on dialysis (p < 0.01), and had a lower Charlson Comorbidity Index (p < 0.01). Serum calcium and intact parathyroid hormone levels were not different between the two groups, while phosphate levels <5.5 mg/dL were observed more frequently in patients not receiving sevelamer (79% vs 61%, p < 0.01). Serum total cholesterol (167 +/- 41 vs 189 +/- 42 mg/dL, p < 0.01) and low density lipoprotein (LDL) cholesterol (90 +/- 34 vs 109 +/- 34 mg/dL, p < 0.01), but not high density lipoprotein cholesterol or triglycerides, were lower in sevelamer-treated patients. Moreover, sevelamer-treated patients displayed a higher serum albumin (38 +/- 5 vs 36 +/- 4 g/L, p < 0.01) and a lower C-reactive protein (4.9 +/- 12.8 vs 8.8 +/- 15.7 mg/L, p < 0.01). Blood bicarbonate levels <22 mmol/L were observed more frequently in patients receiving sevelamer (22% vs 5%, p < 0.01). Logistic regression analysis adjusting by confounding variables confirmed that sevelamer therapy was associated with serum total cholesterol <200 mg/dL [relative risk (RR): 2.77, 95% confidence interval (CI): 1.44 - 5.26, p = 0.002] and blood bicarbonate <22 mmol/L (RR: 8.5, 95% CI: 2.6 - 27.0, p < 0.001), but not with serum phosphate >5.5 mg/dL, calcium-phosphate product >55 mg(2)/dL(2), serum albumin <35 g/L, or C-reactive protein >5 mg/L., Conclusions: This uncontrolled cross-sectional study in peritoneal dialysis patients showed that sevelamer hydrochloride treatment allows an adequate serum phosphate level in about 60% of patients and significantly reduces total and LDL-cholesterol levels. Since this treatment is associated with metabolic acidosis in 22% of patients, we recommend close monitoring of bicarbonate levels in this group of patients until the clinical significance of this result is clarified.

Cell-based methods and the diversity integral criterion (a distance-based technique) are commonly used approaches for assessing the diversity of collections of compounds in terms of space coverage. The main deficiency with cell-based methods is the arbitrariness of cell boundaries which leads to edge effects or cluster artifacts, i.e., situations in which similar molecules separated by a cell boundary yield a higher diversity score than molecules falling within the same cell but which are less similar to each other. We describe a straightforward diversity metric based on quantifying the distance to the center of the bins resulting from partitioning the descriptor space which aims at bypassing these artifacts. The mentioned criteria are compared for the diversity assessment of a set of selections carried out on three combinatorial libraries of different cardinalities. For each method, the influence of its parameters (reference partition and number of points) on their efficacy is examined. Furthermore, the proposed diversity metric is also applied to designing diverse libraries for three test cases. We show that full arrays selected by minimizing the sum of distances to the center of the cells are formed by compounds spaced further apart than selections obtained by maximizing the degree of cell occupancy.

The alpha4beta1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that alpha4beta1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4(+)CD8(+) and CD4(+)CD8(-) adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9(+)/alpha4beta1(+) human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to alpha4beta1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial alpha4beta1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by alpha4beta1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.

Solid-phase synthesis of a small combinatorial library of dihydroceramide analogues as mixtures of erythro and threo diastereomers is described. Some dihydroceramide analogues cause growth arrest and apoptosis in a dose-dependent manner in human alveolar epithelial cells. This activity is likely due to the threo isomers, as evidenced by cellular studies with a pair of diastereomerically pure N-acyldihydrosphingosines. The apoptotic activity reported in this work provides information for the design of new compounds that may provide the basis for the generation of biochemical tools for the study of different pathologies where ceramide and/or dihydroceramide are involved.

The photodynamic process induces cell damage and death by the combined effect of a photosensitizer (PS), visible light, and molecular oxygen, which generate singlet oxygen ((1)O(2)) and other reactive oxygen species that are responsible for cytotoxicity. The most important application of this process with increasing biomedical interest is the photodynamic therapy (PDT) of cancer. In addition to hematoporphyrin-based drugs, 2nd generation PSs with better photochemical properties are now studied using cell cultures, experimental tumors and clinical trials. Porphycene is a structural isomer of porphyrin and constitutes an interesting new class of PS. Porphycene derivatives show higher absorption than porphyrins in the red spectral region (lambda > 600 nm, epsilon > 50000 M-(1)cm(-1)) owing to the lower molecular symmetry. Photophysical and photobiological properties of porphycenes make them excellent candidates as PSs, showing fast uptake and diverse subcellular localizations (mainly membranous organelles). Several tetraalkylporphycenes and the tetraphenyl derivative (TPPo) induce photodamage and cell death in vitro. Photodynamic treatments of cultured tumor cells with TPPo and its palladium(II) complex induce cytoskeletal changes, mitotic blockage, and dose-dependent apoptotic or necrotic cell death. Some pharmacokinetic and phototherapeutic studies on experimental tumors after intravenous or topical application of lipophilic alkyl-substituted porphycene derivatives are known. Taking into account all these features, porphycene PSs should be very useful for PDT of cancer and other biomedical applications.

Background: Receptor rearrangement upon ligand binding (induced-fit) constitutes a complicating factor in structure-based virtual screening, as protein flexibility is only partially included in many high-throughput docking programs. The effect of protein structure in these cases is rarely discussed., Aim: Our objective was to analyze this influence on three aspects of automated ligand docking: (i) the successful reproduction of binding modes; (ii) the performance in binding site detection for a series of initial decoys positioned on the protein surface; and (iii) the extent to which the protein conformation biases the enrichment factors and the diversity in scaffold retrieval of a Virtual Screening experiment., Methods: A fibroblast growth factor receptor (FGFR), for which several structures complexed with different inhibitors are publicly available, was selected as a study case. Besides its biological relevance, FGFR is an interesting target because of the structural changes occurring on ligand binding in receptor tyrosine kinases. Three common scoring functions (AUTODOCK, ChemScore, and GoldScore), under different parameter settings, were employed to dock a set of inhibitors of FGFR into these structures., Results: We show how the choice of one particular protein x-ray structure restricts the docking process to the detection of those compounds that belong to the same chemical series or are similar to the chemotype of the corresponding co-crystallized ligand.

Background: A prospective cohort study was undertaken to compare the rates of the infecting microorganisms of the peritoneal catheter exit-site in three periods of the prophylactic protocol of a peritoneal dialysis program. All patients treated for more than one month on Peritoneal Dialysis were included: Fourty-eight in Period 1 (P1), 48 in Period 2 (P2), and 54 in Period 3 (P3). Each period was of 3 years., Methods: Infection prophylaxis protocol: P1: hydrogen peroxide or povidone iodine and non-occlusive dressing; P2: sterile water (boiled water) instead of antiseptic agents, semi-permeable dressing for taking showers, and nasal mupirocine prophylaxis for Staphylococcus aureus carriers; P3: equal to P2, plus local application of antibiotics in equivocal exit-site for infection and argentic nitrate in granulation tissue., Main Outcome Measure: The rates of catheter infection and microorganisms causing infection were analysed by means of the Poisson regression method. Chi-square and ANOVA when appropriate., Results: The proportion of catheters implanted by nephrologist or surgeon (p<0.01) and modality treatment by CAPD or CCPD (p<0.0001) were significantly different in the three periods, while the Staph. Aureus carrieres was in the limit of significance (p=0.048). Throughout the three periods, a significantly decreasing rate of total (P=0.0035) and acute infections (P<0.001), Staph. aureus (P=0.003) and peritonitis (P=0.0025) were found. The Pseudomonas aer. (P=0.006) and Gram negative Bacteria (P=0.023) decreased significantly in P2. The multiple factor analysis included eight factors: sex, age group, ESRD, DM, catheter implantation (nephrologist, surgeon), modality treatment (CAPD, CCPD), manufacturer and prophylaxis period as possible predictors of the catheter infections, the specific microorganisms and the peritonitis. That analysis revealed the prophylaxis period as the main predictive factor of the improvements found (p<0.02,- p<0.001). In contrast, the Corynebacteria spp. increased significantly (P=0.008) throughout the three periods. One half of the Corynebacteria in each period could be considered colonisers. The other half caused true infections, but not one of those episodes required catheter intervention. The non-diphtheria Corynebacteria increase was found related with the continuous cycling Peritoneal Dialysis treatment in multiple factor analysis (p=0.0023) and in the proportion analysis (P=0.039, c2)., Conclusion: The progressive protocol applied obtained good results, without the continued use of local antiseptics or antibiotics at the exit-site. However, the non-diphtheria Corynebacteria sp. infection increment favours the consideration of an antiseptic agent for the exit-site care.

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.

The synthesis of novel N-acylethanolamines and their use as inhibitors of the aCDase is reported here. The compounds are either 2-oxooctanamides or oleamides of sphingosine analogs featuring a 3-hydroxy-4,5-hexadecenyl tail replaced by ether or thioether moieties. It appears that, within the 2-oxooctanamide family, the C3-OH group of the sphingosine molecule is required for inhibition both in vitro and in cultured cells. Furthermore, although the (E)-4 double bond is not essential for inhibitory activity, the (E) configuration is required, since the analogue with a (Z)-4 unsaturation was not inhibitory. None of the oleamides inhibited the aCDase in vitro. Conversely, with the exception of N-oleoylethanolamine and its analogs with S-decyl and S-hexadecyl substituents, all the synthesized oleamides inhibited the aCDase in cultured cells, although with a relatively low potency. We conclude that novel aCDase inhibitors can evolve from N-acylation of sphingoid bases with electron deficient-acyl groups. In contrast, chemical modification of the N-oleoylsphingosine backbone does not seem to offer an appropriate strategy to obtain aCDase inhibitors.

Both the radical anion and radical cation of 2,7,12,17-tetraphenylporphycene and of its palladium(II) complex have been produced by photochemical methods. Their electronic absorption spectrum and decay kinetics have been characterised by global analysis of the time-absorbance-wavelength matrix. The results provide deeper insight on the role of these radical ion species in the photodynamic activity of tetraphenylporphycenes.

The need for new photodynamic-therapy photosensitizers has stimulated the search of new families of compounds absorbing strongly in the 700-900 nm range, the region where tissue is most transparent to radiation capable to induce the photodynamic effect. Using computational chemistry techniques, 3,6,13,16-tetraazaporphycenes were previously identified as interesting target candidates. This work reports on the photophysical and electrochemical properties of selected members of this new family of macrocycles. Compared to porphycenes, the tetra-aza counterparts show stronger absorption in the near-infrared, lower-lying singlet and triplet excited states, and substantially larger internal conversion quantum yield (Phi(IC) = 0.93). Energy transfer to oxygen is observed, which results in the formation of the cytotoxic species singlet oxygen. The process is found to be reversible, consistent with a triplet-energy value close to that of singlet oxygen.

A new Suzuki-based strategy for the synthesis of 4,4'-diaryl (or heteroaryl)-substituted 2,2'-bipyrroles (10), precursors of 2,7,12,17-tetraaryl (or heteroaryl)-substituted porphycenes, is described. Bromination of the previously described diethyl 4H,5H-thieno[3,2-b:4,5-b']dipyrrole-3,6-dicarboxylate afforded dibromo compound 19, which is the key intermediate of such strategy.

A compilation of combinatorial chemistry techniques applied to anti-HIV drug development is presented in this review. This synthetic strategy together with high throughput screening assays has allowed the discovery and optimization of novel lead anti-HIV compounds.

Melanoma cells express the chemokine receptor CXCR4, which confers invasive signals on binding to its ligand CXCL12. We show here that knocking down membrane-type matrix metalloproteinase (MT1-MMP) expression translates into a blockade of invasion across reconstituted basement membranes and type I collagen gels in response to CXCL12, which is the result of lack of MMP-2 activation. Interference with MMP-2 expression further confirms its important role during this invasion. Vav proteins are guanine-nucleotide exchange factors for Rho GTPases that regulate actin dynamics and gene expression. We show that melanoma cells express Vav1 and Vav2, which are activated by CXCL12 involving Jak activity. Blocking Vav expression by RNA interference results in impaired activation of Rac and Rho by CXCL12 and in a remarkable inhibition of CXCL12-promoted invasion. Importantly, up-regulation of MT1-MMP expression by CXCL12, a mechanism contributing to melanoma cell invasion, is blocked by knocking down Vav expression or by inhibiting Jak. Together, these data indicate that activation of Jak/Vav/Rho GTPase pathway by CXCL12 is a key signaling event for MT1-MMP/MMP-2-dependent melanoma cell invasion.

Malignant plasma cells in multiple myeloma home to the bone marrow (BM), accumulate in different niches and, in late disease, migrate from the BM into blood. These migratory events involve cell trafficking across extracellular matrix (ECM)-rich basement membranes and interstitial tissues. Metalloproteinases (MMP) degrade ECM and facilitate tumour cell invasion. The chemokine CXCL12 is expressed in the BM, and it was previously shown that it triggers myeloma cell migration and activation. In the present work we show that CXCL12 promotes myeloma cell invasion across Matrigel-reconstituted basement membranes and type I collagen gels. MMP-9 activity was required for invasion through Matrigel towards CXCL12, whereas TIMP-1, a MMP-9 inhibitor that we found to be expressed by myeloma and BM stromal cells, impaired the invasion. In addition, we show that the membrane-bound MT1-MMP metalloproteinase is expressed by myeloma cells and contributes to CXCL12-promoted myeloma cell invasion across Matrigel. Increase in MT1-MMP expression, as well as induction of its membrane polarization by CXCL12 in myeloma cells, might represent potential mechanisms contributing to this invasion. CXCL12-promoted invasion across type I collagen involved metalloproteinases different from MT1-MMP. These data indicate that CXCL12 could contribute to myeloma cell trafficking in the BM involving MMP-9 and MT1-MMP activities., (Copyright 2005 Pathological Society of Great Britain and Ireland.)

The small GTPase Rac contributes to regulation of cytoskeletal rearrangement during chemokine-induced lymphocyte adhesion and migration in a multi-step process that is very precisely coordinated. Chimaerins are Rac1-specific GTPase-activating proteins of unknown biological function, which have a canonical diacylglycerol C1-binding domain. Here we demonstrate endogenous expression of beta2-chimaerin in T lymphocytes and study the functional role of this protein in phorbol ester and chemokine (CXCL12)-regulated T-cell responses. We used green fluorescent protein-tagged beta2-chimaerin and phorbol ester stimulation to investigate changes in protein localization in living lymphocytes. Our results demonstrate that active Rac cooperates with C1-dependent phorbol ester binding to induce sustained GFP-beta2-chimaerin localization to the membrane. Subcellular distribution of GFP beta2-chimaerin in living cells showed no major changes following CXCL12 stimulation. Nonetheless Rac1-GTP levels were severely inhibited in GFP-beta2-chimaerin-expressing cells, which displayed reduced CXCL12-induced integrin-dependent adhesion and spreading. This effect was dependent on chimaerin GTPase-activating protein function and required diacylglycerol generation. Whereas beta2-chimaerin overexpression decreased static adhesion, it enhanced CXCL12-dependent migration via receptor-dependent diacylglycerol production. These studies demonstrate that beta2-chimaerin provides a novel, diacylglycerol-dependent mechanism for Rac regulation in T cells and suggest a functional role for this protein in Rac-mediated cytoskeletal remodeling.

Introduction: Despite the interest generated by the increasing number of studies that measure Quality of Life among patients and caregivers, few of these studies measure the caregivers burden in Peritoneal Dialysis (PD)., Objective: The main target of this study was to create a burden measure questionnaire applicable amongst caregivers of PD patients., Inclusion Criteria: 1) Patients had to be in PD treatment for more than 3 months; 2) Patients had to receive help with the PD treatment from a caregiver. The study was divided into 3 phases: 1st) design and use of the initial questionnaire; 2nd) a test-retest on a modified scale; and 3rd) to provide the questionnaire-3 to two collaborative centres with similar PD programs. Four groups of caregivers were established: A1:23, A2:17, B:7 and C:16 caregivers. We applied 5 scales (5): 1--Patient Dependence on caregiver, from caregivers' view (D); 2--Complete caregiver burden (CB), including 12 items which measure the caregivers' subjective burden, 3--Reduced caregiver burden (RB), as the one before but with only 8 items, 4--Repercussions on the caregiver (R), which measures objective burden; 5--Specific PD tasks (ST), a scale that measures the effort the tasks implied in the PD treatment represent for the caregiver., Results: We studied 63 caregivers (table I): mean age: 53.43 (SD = 12.3); Sex: Females: 86.4%, Males: 13.6%, corresponding to 63 patients: mean age: 59.79 (SD = 15.9); Sex: Males: 80.3%, Females: 19.7%. Valuable results for reliability, unidimensionality, and discrimination were obtained in the 1st and 2nd phases, except for burden scale which was compound of two factors; then one of those factors was suppressed. In the 3rd phase, ANOVA did not show any differences between centres (table II). Consequently, all caregivers could be analysed together. Reliability results for each one of the third phase scales (table III) were: D: Cronbach alpha = 0,886; CB: alpha = 0,894; RB: alpha = 0,857; R: alpha = 0,892; ST: alpha = 0,62. Although the ST scale obtained an acceptable reliability, it was suppressed in the 3rd phase due to the low correlation with other scales and the fact that it was not applicable to all caregivers. Finally, a direct correlation was found between third phase scales (table IV): D-RB: r = 0.502, p < or = 0.001; D-R: r = 0.599, p < or = 0.001; RB-R: r = 0.775, p < or = 0.001. We must headlight that both Burden scales, and the Repercussion scale, obtained a direct correlation with the Dependency scale., Conclusion: A questionnaire has been created to measure burden and repercussions on caregivers of peritoneal dialysis patients. It can already be applied, as requirements of both reliability and validity are fulfilled. This questionnaire can be a useful tool to prevent caregivers' burnout.

Among the known non-benzodiazepinic hypnotic drugs acting on the alpha1 subunit of the GABA-A receptor, Zolpidem, Zaleplon and Indiplon have showed high affinity and selectivity. Following a design methodology including pharmacophoric requirements and ADME-predicted properties, we have synthesized a library of 3-amino-4,5-dihydro-1H-pyrazolo[3,4-b]pyridin-6(7H)-ones and their N1-alkyl derivatives as new scaffolds for designing non-benzodiazepine BZ receptor ligands.

The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.

Multiple myeloma is characterized by the accumulation of terminally differentiated B cells in the bone marrow, due to increased proliferation and restricted apoptosis of the myelomatous clone. Here we have studied the participation of a novel mitogen-activated protein kinase (MAPK) route, the extracellular signal-regulated kinase 5 (Erk5) pathway, in the regulation of myeloma cell proliferation and apoptosis. Erk5 was expressed in cells isolated from patients and in myeloma cell lines. The myeloma growth factor interleukin 6 (IL-6) activated Erk5, and this activation was independent of Ras and Src. Expression of a dominant-negative form of Erk5 restricted the proliferation of myeloma cells and inhibited IL-6-dependent cell duplication. This dominant-negative form also sensitized myeloma cells to the proapoptotic action of dexamethasone and PS341. The latter compound caused a profound decrease in the amount of endogenous Erk5 and was less effective in inducing apoptosis when the level of Erk5 was increased by transfection of Erk5. These results place the Erk5 route as a new regulatory signaling pathway that affects multiple myeloma proliferation and apoptosis.

Heterocyclic demonstration libraries for agrochemical screening were prepared from the common intermediates 2-methoxy-6-oxo-1,4,5,6-tetrahydropyridine-3-carbonitriles (1), using standard solution-phase techniques. A total of 18 screening libraries were prepared in good to excellent yields. Several members of these libraries were active in the first level of agrochemical screening, especially in the fungicide screen.

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is expressed by bone marrow (BM) stromal cells and plays key roles in cell homing to and retention into the bone marrow. In multiple myeloma, blood-borne malignant plasma cells home to the BM and accumulate in contact with stromal cells, implicating myeloma cell migration across endothelium. Myeloma cells express the SDF-1alpha receptor CXCR4, as well as the integrin alpha4beta1, which mediates their attachment to BM stroma. We show here that SDF-1alpha promotes transendothelial migration of purified BM myeloma cells and myeloma-derived NCI-H929 cells, involving a transient upregulation of alpha4beta1-dependent cell adhesion to the endothelium. Characterization of intracellular signaling pathways involved in the modulation by SDF-1alpha of alpha4beta1-mediated myeloma cell adhesion revealed that intracellular cAMP amounts associated with the activation of protein kinase A play key roles in this modulation. Furthermore, a functional link between cAMP actions on the dynamics of actin cytoskeleton, RhoA activation, and alpha4beta1-dependent cell adhesion in response to SDF-1alpha has been found. The regulation of alpha4beta1-mediated myeloma cell adhesion by SDF-1alpha could play key roles during myeloma cell homing into and trafficking inside the BM, and characterization of the molecular events involved in SDF-1alpha-activated modulation of this adhesion will contribute to a better understanding of mechanisms participating in cell migration.

Tissue invasion by tumor cells involves their migration across basement membranes through activation of extracellular matrix degradation and cell motility mechanisms. Chemokines binding to their receptors provide chemotactic cues guiding cells to specific tissues and organs; they therefore could potentially participate in tumor cell dissemination. Melanoma cells express CXCR4, the receptor for the chemokine stromal cell-derived factor-1alpha (SDF-1alpha). Using Matrigel as a model, we show that SDF-1alpha promotes invasion of melanoma cells across basement membranes. Stimulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) activity by SDF-1alpha was necessary for invasion, involving at least up-regulation in the expression of this metalloproteinase, as detected in the highly metastatic BLM melanoma cell line. Moreover, SDF-1alpha triggered the activation of the GTPases RhoA, Rac1, and Cdc42 on BLM cells, and expression of dominant-negative forms of RhoA and Rac1, but not Cdc42, substantially impaired the invasion of transfectants in response to SDF-1alpha, as well as the increase in MT1-MMP expression. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-beta1, a Matrigel component, whereas anti-transforming growth factor-beta antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1alpha. The identification of SDF-1alpha as a potential stimulatory molecule for MT1-MMP as well as for RhoA and Rac1 activities during melanoma cell invasion, associated with an up-regulation in CXCR4 expression by interaction with basement membrane factors, could contribute to better knowledge of mechanisms stimulating melanoma cell dissemination.

Two new solid-phase syntheses of substituted pyrazoles are described. The first includes supporting an o-hydroxyacetophenone on Merrifield resin, Vilsmeier-Haack formylation on the methyl group and cyclization with a substituted hydrazine to afford a pyrazole ring with two diversity centers. The second starts from o-hydroxyacetophenone supported on Wang resin, which undergoes a Claisen condensation with a carboxylic acid ester to yield a 1,3-dicarbonyl compound that cyclizes to a pyrazole using a hydrazine. Both methods have been used to synthesize two small pyrazole libraries.

Chemokine stromal cell-derived factor-1 (SDF-1) is expressed by bone marrow (BM) stromal cells and plays key roles in BM cell migration. Modulation of its expression could affect the migratory capacity of cells trafficking the BM, such as hematopoietic progenitor and leukemic cells. Transforming growth factor-beta1 (TGF-beta1) is present in the BM environment and constitutes a pivotal molecule controlling BM cell proliferation and differentiation. We used the BM stromal cell line MS-5 as a model to investigate whether SDF-1 expression constitutes a target for TGF-beta1 regulation and its functional consequences. We show here that TGF-beta1 down-regulates SDF-1 expression, both at the mRNA level, involving a decrease in transcriptional efficiency, and at the protein level, as detected in lysates and supernatants from MS-5 cells. Reduction of SDF-1 in supernatants from TGF-beta1-treated MS-5 cells correlated with decreased, SDF-1-dependent, chemotactic, and transendothelial migratory responses of the BM model cell lines NCI-H929 and Mo7e compared with their responses to supernatants from untreated MS-5 cells. In addition, supernatants from TGF-beta1-exposed MS-5 cells had substantially lower efficiency in promoting integrin alpha4beta1-mediated adhesion of NCI-H929 and Mo7e cells to soluble vascular cell adhesion molecule-1 (sVCAM-1) and CS-1/fibronectin than their untreated counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1-dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to sVCAM-1 when supernatants from TGF-beta1-treated MS-5 cells were used compared with supernatants from untreated cells. These data indicate that TGF-beta1-controlled reduction in SDF-1 expression influences BM cell migration and adhesion, which could affect the motility of cells trafficking the bone marrow.

A large virtual library of 125 396 HEPT analogues, built by combining all fragments present in the published 180-compound HEPT family, has been studied in terms of diversity criteria and the goodness of the 11 available standard diversity selection methods analyzed. All the algorithms under study, except Cell-based Density, have rank above a random selection of compounds, with Optimum and Standard Deviation based Binning and Cell-based Fraction algorithms being the best choices. Furthermore, analysis of the actually tested compounds has been performed to compare the traditional drug discovery methodology versus a rational selection of combinatorial libraries approach.

A high yield multicomponent reaction providing multifunctionalized pyrido[2,3-d]pyrimidines with up to four diversity centers in a one-pot microwave-assisted cyclocondensation of alpha,beta-unsaturated esters, amidine systems and malononitrile (or ethyl cyanoacetate) is described.

We have implemented and adapted in Pralins (Program for Rational Analysis of Libraries in silico), the most popular sparse (cherry picking) and full array (sublibrary) selection algorithms: hierarchical clustering, k-means clustering, Optimum Binning, Jarvis Patrick, Pral-SE (partitioning techniques) and MaxSum, MaxMin, MaxMin averaged, DN2, CTD (distance-based methods). We have validated the program with an already synthesized three-component combinatorial library of FXR partial agonists characterized by standard computational chemistry descriptors as case study. This has let us analyze the goodness of both the partitioning techniques for space division and all the selection methodologies with respect to representativity in terms of population and space coverage for different selection sizes. Within the chemical space analyzed, both hierarchical clustering and Optimum Binning division strategies are found to be the most advantageous reference space divisions to be used in the subsequent population and space coverage studies. Complete hierarchical clustering appears also to be the preferred selection methodology for both sparse and full array problems. The full array restriction fulfillment can easily be overcome by convenient optimization algorithms that allow optimal reagent selection preserving > 90% of the population coverage.

The alpha4 integrins (alpha4beta1 and alpha4beta7) are cell surface heterodimers expressed mostly on leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature of alpha4 integrins is that their adhesive activity can be subjected to rapid modulation during the process of cell migration. Herein, we show that transforming growth factor-beta1 (TGF-beta1) rapidly (0.5-5 min) and transiently up-regulated alpha4 integrin-dependent adhesion of different human leukocyte cell lines and human peripheral blood lymphocytes (PBLs) to their ligands vascular cell adhesion molecule-1 (VCAM-1) and connecting segment-1/fibronectin. In addition, TGF-beta1 enhanced the alpha4 integrin-mediated adhesion of PBLs to tumor necrosis factor-alpha-treated human umbilical vein endothelial cells, indicating the stimulation of alpha4beta1/VCAM-1 interaction. Although TGF-beta1 rapidly activated the small GTPase RhoA and the p38 mitogen-activated protein kinase, enhanced adhesion did not require activation of both signaling molecules. Instead, polymerization of actin cytoskeleton triggered by TGF-beta1 was necessary for alpha4 integrin-dependent up-regulated adhesion, and elevation of intracellular cAMP opposed this up-regulation. Moreover, TGF-beta1 further increased cell adhesion mediated by alpha4 integrins in response to the chemokine stromal cell-derived factor-1alpha. These data suggest that TGF-beta1 can potentially contribute to cell migration by dynamically regulating cell adhesion mediated by alpha4 integrins.

An efficient solid-phase synthesis of 2-substituted 4-aminopyrido[2,3-d]pyrimidines 15 is reported. The procedure started by solid supporting a p-hydroxybenzaldehyde 8 to the Wang resin by using the Mitsunobu protocol. The resulting aldehyde 17 was treated with a substituted acid methyl malonate 10 to afford the corresponding alpha, beta-unsaturated ester 18, which was converted to the Michael adduct 21 by reaction with malononitrile. Cyclization of 21 with an amidine system 13 yielded the solid supported pyridopyrimidine 22, which afforded the corresponding 2-substituted 4-aminopyrido[2,3-d]pyrimidine 15 upon treatment with TFA:DCM. Compounds 15 present three diversity centers R1, R2 and R3. Having validated the chemistry on solid support, a 32-membered combinatorial library was obtained using this protocol.

During hematopoiesis in the bone marrow (BM), hematopoietic progenitor cells (HPC) proliferate and differentiate in intimate contact with BM stromal cells in a highly regulated interplay among cytokines, chemokines, and adhesion molecules. Using the mouse BM stromal cell line MS-5 as immunogen, we raised monoclonal antibodies (mAb) against surface proteins that could participate in interactions between HPC and stroma. Flow cytometry and immunodepletion analysis indicated that two antibodies, MA-8 and MA-2, recognized CD44. Although MA-8 and another anti-CD44 antibody, IM7.8.1, blocked adhesion of hematopoietic cells to hyaluronate, adhesion to BM stroma was notably increased by these mAb, suggesting minor roles for CD44/hyaluronate interaction and the triggering of additional adhesion pathways upon CD44 engagement. The presence of MA-8 in Dexter-type long-term BM cultures (LTBMC) only slightly inhibited production of myeloid cells, and the number of myeloid progenitors (measured as CFU-GM) in the supernatants of LTBMC performed with MA-8 was similar to those carried out with control antibodies. Instead, IM7.8.1 completely blocked myelopoiesis, whereas an anti-alpha 4 integrin mAb increased CFU-GM amounts. In summary, two anti-CD44 antibodies with similar effects in cell adhesion showed notable differences in myelopoietic cultures, indicating the structural and functional complexity of the CD44 molecule.

The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.

The preparation in multigram scale of two metabolites 3-(3,5-dichloro-4-methyl-benzoylamino)-2-hydroxy-3-methyl-pentanoic acid and 3-(3,5-dichloro-benzoylamino)-3-methyl-2-oxo-butyric acid isolated from soil treated with either Zoxium fungicide or Kerb herbicide was efficiently accomplished using a common 5-step synthetic process starting from easily available raw materials.

Introduction: Peripheral neuropathy is usually one of the initial symptoms of necrotizing vasculitis. Early diagnosis is essential for good prognosis., Objective: To determine which parameters are useful for early diagnosis and selection of tissue for biopsy., Patients and Methods: We analyzed the clinical, biological, electromyographic and anatomopathological characteristics of 26 patients with necrotizing vasculitis and peripheral neuropathy., Results: Twelve patients had panarteritis nodosa, three Churg Strauss syndrome, two Wegener s syndrome, two disseminated lupus erythematosis, one sarcoidosis and one Walderstrom s macroglobulinemia. Fifteen patients had multineuritis and the remainder distal mixed polyneuropathy which was symmetrical in three cases. In five cases biopsy was normal. Sural nerve biopsy showed the diagnosis to be correct in 20% of the patients. However, when this was done on a neurophysiologically affected nerve, the tissue was seen to be altered in 61%. Biopsy of the gastrocnemius increased the degree of usefulness to 73% when electromyographic anomalies were detected in this muscle., Conclusions: Neurophysiological study is essential for detection of alterations in patients clinically suspected of having necrotizing vasculitis, even in cases where there is apparently no neuropathy. We recommend biopsy of the gastrocnemius muscle as the first choice in cases where sural nerve neurography is found to be normal. If the sural nerve is not normal, it is the site of choice for biopsy, and muscle biopsy is the site of second choice. When both biopsies are normal, renal biopsy should be considered to establish the diagnosis, as a third alternative.

We have examined the role of the small GTPase Rho and its downstream effector, the Rho-associated kinase (ROCK), in the control of the adhesive and signaling function of the lymphocyte function-associated antigen-1 (LFA-1) integrin in human T-lymphocytes. Inhibition of Rho (either by treatment with C3-exoenzyme or transfection with a dominant-negative form of Rho (N19Rho)) or ROCK (by treatment with Y-27632) results in the following: (a) partial disorganization and aggregation of cortical filamentous actin (F-actin); (b) induction of LFA-1-mediated cellular adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) through a mechanism involving clustering of LFA-1 molecules, rather than alterations in the level of expression or in the affinity state of this integrin; and (c) induction of cellular polarization and activation of the tyrosine kinase PYK2. Transfection of T-cells with a constitutively active form of Rho (V14Rho) blocks the clustering of LFA-1 on the membrane and the LFA-1-mediated activation of PYK2. Importantly, the activation of PYK2 caused by inhibition of Rho or ROCK takes place only when the T-cells are plated onto ICAM-1 but not when they are either prevented from interacting with ICAM-1 with anti-LFA-1 blocking antibodies or when they are plated on the nonspecific poly-l-lysine substrate. These results indicate that the small GTPase Rho regulates the tyrosine kinase PYK2 in T-cells through the F-actin-mediated control of the activity of the integrin LFA-1. These findings represent a novel paradigm for the regulation of the activity of a cytoplasmic tyrosine kinase by the small GTPase Rho.