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PubMed2004

The CD11/CD18 (beta2) integrins modulate neutrophil caspase activation and survival following TNF-alpha or endotoxin induced transendothelial migration.

Neutrophils (PMN) are short-lived cells but their survival is often prolonged in inflammation. The beta2 (CD11/CD18) integrins are involved in PMN migration into inflammation but their role in PMN survival is not well understood. We investigated the role of beta2 integrins in PMN caspase activation, a key enzyme cascade in apoptosis. After 20 h, caspase activation (Western blotting) was markedly decreased in PMN cultured on fibrinogen, a ligand for Mac-1 (CD11b/CD18), but not on fibronectin or albumin. In the presence of TNF-alpha or endotoxin (LPS), blockade of CD18 (beta2 chain) with mAb markedly increased caspase activation in PMN on fibrinogen. PMN which migrated through endothelium in vitro in response to TNF-alpha, LPS, IL-1alpha, IL-8 or C5a contained 58% fewer active caspase positive PMN after 20 h than non-migrated PMN remaining on the endothelium. When beta2 (CD18) integrin or lymphocyte function antigen (LFA)-1 (CD11a) plus Mac1 (CD11b) were blocked by mAb (intact or Fab'), the proportion of migrated PMN (but not of non-migrated PMN) with active caspases was significantly increased (2-4-fold) and this was associated with accelerated PMN apoptosis and death. Thus, engagement of ligands on extracellular matrix and endothelium by the beta2 integrins Mac-1 and LFA-1 plays a role in delaying apoptosis in PMN recruited in response to LPS and TNF-alpha. Inhibition of beta2 integrin function may not only inhibit PMN infiltration, but also accelerate PMN clearance from inflamed tissue.

Apoptosis↗

PubMed2004

Population dynamics of Aedes aegypti from a dengue hyperendemic urban setting in Colombia.

This study evaluated if the Aedes aegypti population in the city of Cali, Colombia was composed of genetically distinct local populations with different levels of insecticide resistance and dengue vector competence. Insecticide resistance was assayed biochemically and was associated with varying levels of mixed-function oxidases and non-specific esterases. The genes encoding those enzymes were under selective pressure from insecticides used to suppress Ae. aegypti populations. Vector competence showed heterogeneity among the vector populations ranging from 19% to 60%. Population genetic analysis of random amplified polymorphic DNA-polymerase chain reaction products, expressed as genetic distance, Wright's F(st), and migration rate (Nm), demonstrated moderate genetic differentiation among Ae. aegypti from four sites (F(st) = 0.085). The results from all characteristics evaluated in the study demonstrated spatial and temporal variation between Ae. aegypti populations. At any specific time, the local populations of Ae. aegypti were genetically differentiated and unique with respect to insecticide resistance and vector competence. Both characteristics changed independently.

Aedes↗

PubMed2005

Decomposition of protonated noradrenaline and normetanephrine assisted by NH2 migration studied by electrospray tandem mass spectrometry and molecular orbital calculations.

As part of a research program on neurotransmitters in a biological fluid, the fragmentations characterising catecholamines protonated under electrospray ionisation (ESI) conditions, under low collision energy in a triple-quadrupole mass spectrometer, were investigated. The decompositions of protonated noradrenaline (VH) and normetanephrine (VIH) were studied. Both precursor ions eliminate first H2O at very low collision energy, and the fragmentations of [MH-H2O]+ occur at higher collision energy. The breakdown graphs of [MH-H2O]+ ions, with collision energy varying from 0-40 eV in the laboratory frame, are presented. [VIH-H2O]+ ions lose competitively NH3 and CH3OH. For [VH-H2O]+ the loss of NH3 is dominant while H2O is eliminated at very low abundance at all collision energies. All of these secondary fragmentations are followed at higher collision energies by elimination of CO. These fragmentations are interpreted by means of ab initio calculations up to the B3LYP/6-311+G(2d,2p) level of theory. The elimination of H2O requires first the isomerisation of N-protonated forms, chosen as energy references, to O-protonated forms. The isomerisation barriers are calculated to be lower than 81 kJ/mol above the N-protonated forms. The elimination of NH3 from [MH-H2O]+ requires first the migration, via a cyclisation, of the amine function from the linear chain to the aromatic ring in order to prevent the formation of unstable disubstituted carbocations in the ring. The barriers associated with the loss of NH3 are located 220 and 233 kJ/mol above VH and 219 kJ/mol above VIH. The energy barrier for the loss of ROH is located 236 and 228 kJ/mol above VH and VIH, respectively. The absence of ions corresponding to [VH-2H2O]+ is due to a parasitic mechanism with an activation barrier lower than 236 kJ/mol that leads to a stable species unable to fragment, thus preventing the second loss of H2O. Losses of CO following the secondary fragmentations involve activation barriers higher than 330 kJ/mol.

Algorithms↗

PubMed2000

The Drosophila Lissencephaly1 (DLis1) gene is required for nuclear migration.

Nuclear movement is critical for several developmental processes in eukaryotes. Drosophila oogenesis provides a paradigmatic example in which localization of the nucleus generates a source of cellular asymmetry that is used in patterning both the anterior-posterior and the dorsal-ventral axes of the oocyte. In this study we show that mutations in the Drosophila Lissencephaly1 (DLis1) gene result in partial ventralization of the eggshell. DLis1 mutations affect the localization of gurken mRNA and protein in the oocyte. These defects are correlated with incorrect positioning of the oocyte nucleus, suggesting that DLis1 is required for nuclear migration. DLis1 shows significant sequence conservation across the evolutionary spectrum. Fungal cognates of DLis1 are involved in nuclear migration while homologs in humans and mice are implicated in neuronal migration. DLis1 shows genetic interactions with the Glued and Dynein heavy chain subunits of the dynein/dynactin complex, supporting the idea that the Lis1 family of proteins plays a role in microtubule motor-based nuclear motility.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1988

Highly glycosylated PDGF-like molecule secreted by simian sarcoma virus-transformed cells.

Antiserum to human platelet-derived growth factor (PDGF) recognized a simian sarcoma virus transformation-specific glycopeptide, now termed gp200sis, thereby establishing an immunological relationship between PDGF and this highly glycosylated molecule. The same antibodies as well as an antiserum against SSV-NP cells reacted with isolated gp200sis after immunoprecipitation, SDS-PAGE, and electroelution. In analogy to PDGF, the gp200sis protein backbone is shown here to consist of disulfide-linked polypeptide chains. On SDS-PAGE under nonreducing conditions, the deglycosylated molecule migrated as two dimers with molecular weights of 26 and 28 kDa, respectively. Preliminary functional studies indicate that SSV nonproducer cells secrete high-molecular-weight mitogens (greater than 150 kDa) that are specific for SSV-induced transformation. We suggest that gp200sis acts as a PDGF-like growth factor.

Cell Transformation, Viral↗

PubMed1992

Comparative serological reactivity of Taenia crassiceps, Taenia solium and Taenia saginata metacestode neutral glycolipids to infection serum from Taenia crassiceps-infected mice.

A comparative survey was undertaken of the neutral fraction glycolipids from the metacestodes of 3 taeniid species, Taenia crassiceps, Taenia solium and Taenia saginata, to determine their chemical and serological staining patterns on separation by thin-layer chromatography. The orcinol-positive patterns of T. solium and T. saginata metacestodes exhibited a closer superficial resemblance to each other than to T. crassiceps or T. saginata adults. A comparison of component migration properties against standards of known structure indicated the main oligosaccharide chains to be mono-, di-, tri- and tetrasaccharides; however, in T. solium this was extended to at least a heptasaccharide. The multiple banding characteristic of each component is a consequence of lipid moiety heterogeneity. Serologically, the patterns of the 3 taeniid species neutral fraction glycolipids showed virtually the same immunological reactivity towards mouse normal serum, infection serum and a monospecific, polyclonal antibody directed against the trisaccharide component of T. crassiceps. The latter antibody was isolated from mouse infection serum by affinity chromatography on a column of glycolipid-bound octyl-Sepharose CL-4B. Immunochemically, the major common epitope expressed by the neutral fraction glycolipids of the 3 taeniid species is the same or very similar to the glycosphingolipid, neogalatriaosyl ceramide derived from the marine mollusc Turbo cornutus (Gal(beta 1-6) Gal(beta 1-6) Gal(beta 1-1)Cer). Host tissue neutral fraction glycolipids, porcine muscle and bovine muscle, as well as human spleen, were not immunoreactive.

Animals↗

PubMed1990

Uncoupling of muscle-specific protein expression in myocyte x myoblast heterokaryons.

The inducibility of several rat skeletal muscle proteins was examined in heterokaryons formed by fusing differentiated chick myocytes to undifferentiated rat myoblasts. Chicken and rat proteins were distinguished using species-specific antibodies or by their different migrations in polyacrylamide or agarose gels. Both rat skeletal myosin light chain 1 and rat alpha-tropomyosin were induced in the heterokaryons. In contrast, neither rat acetylcholine receptors nor creatine kinase could be detected. These results suggest that chick myocytes may contain quantities of regulatory factors that are sufficient for the activation of some but not all of these rat muscle-specific proteins within the cellular context of the heterokaryon.

Animals↗

PubMed1996

Structural and immunochemical studies on the lipopolysaccharide of the 'T-antigen'-containing mutant Proteus mirabilis R14/1959.

In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained D-glucose, D-galacturonic acid (D-GalA), and D-GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42. The importance of a D-GalA(L-Lys)-containing epitope, most likely present in the core region of LPS, and of GalA present in the T-antigen chain in manifesting the serological specificity of P. mirabilis R14/1959 were revealed using rabbit polyclonal homologous and heterologous R- and O-specific antisera and the appropriate antigens, including synthetic antigens which represent partial structures of various Proteus LPS.

Animals↗

PubMed2003

Essential role of glycosaminoglycans in Fgf signaling during mouse gastrulation.

In vitro studies have suggested that proteoglycans facilitate signaling by mammalian growth factors, but genetic evidence supporting this role has been lacking. Here, we characterize the ENU-induced mutation lazy mesoderm (lzme), which disrupts the single mouse gene encoding UDP-glucose dehydrogenase (Ugdh), an enzyme required for the synthesis of the glycosaminoglycan (GAG) side chains of proteoglycans. lzme mutants arrest during gastrulation with defects in migration of mesoderm and endoderm, a phenotype similar to that of mutants in the fibroblast growth factor (Fgf) pathway. Analysis of the expression of molecular markers indicates that Fgf signaling is blocked in lzme mutant embryos. In contrast, signaling by the growth factors Nodal and Wnt3, which are also essential during mouse gastrulation, appears to be normal in lzme embryos. The results demonstrate that proteoglycans are required during mouse gastrulation specifically to promote Fgf signaling.

Animals↗

PubMed1996

Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats.

The 5/6 nephrectomy model is used to study pathogenetic mechanisms underlying chronic renal failure. We previously demonstrated that increased mesangial cell proliferation and glomerular PDGF B-chain expression precede glomerulosclerosis in this model. In the present study we have assessed the concomitant changes in the cortical tubulointerstitium. A wave of tubular and interstitial cell proliferation (as determined by immunostaining for PCNA) occurred at week 1 after 5/6 nephrectomy. This wave preceded the peak glomerular cell proliferation by one week. Tubulointerstitial cell proliferation decreased thereafter and reached control values by week 10. In situ hybridization and immunostaining for PDGF B-chain and beta-receptor in sham-operated controls showed labeling of distal tubules and collecting ducts, while no signal was present in the interstitium. PDGF B-chain mRNA and protein expression was markedly increased in tubules at weeks 2 and 4 after 5/6 nephrectomy and in the interstitium (particularly in areas of inflammatory infiltrates) at weeks 2 to 10. Similar changes occurred with PDGF receptor beta-subunit immunostaining. Interstitial expression of desmin and alpha-smooth muscle actin (markers of myofibroblasts) progressively increased after week 1. Interstitial influx of monocytes/macrophages with focal accentuation started at week 2. Counts of lymphocytes, neutrophils and platelets showed only minor changes. In parallel to the monocyte/macrophage influx, progressive interstitial accumulation of collagens I and IV, laminin, and fibronectin occurred. All of these changes were correlated with the increase in serum creatinine, proteinuria and an index of tubulointerstitial damage. We conclude that tubulointerstitial changes after 5/6 nephrectomy show similarities with those observed in the glomeruli. Tubular and interstitial overexpression of PDGF B-chain and its receptor may play a role in mediating fibroblast migration and/or proliferation in areas of tubulointerstitial injury.

Actins↗

PubMed2001

Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival.

Peritoneal dissemination is the most frequent progression pathway of ovarian cancer and is therefore a key step to improve the prognosis. NK4, a large part of the alpha-chain of hepatocyte growth factor, is known to inhibit cancer cell migration. To characterize the function of NK4 and investigate its potential role in gene therapy of ovarian cancer, we introduced NK4 cDNA to an ovarian cancer cell line HRA and investigated its effects both in vitro and in vivo. HRA cells were transfected with either NK4 or luciferase-expression plasmids. After selection, NK4-expressing HRA cells (HRA/NK4) and the control cells (HRA/LUC) were obtained. NK4 was detected in the culture supernatant of HRA/NK4 by Western analysis. Migration capabilities of the cells were evaluated in vitro by scratch wound healing assay. The number of migrated cells was significantly smaller in the HRA/NK4 cultures than that in the control cultures (HRA or HRA/LUC). Also, the culture supernatant of HRA/NK4 significantly suppressed migration of control cells. This suppressive effect was observed when NK4-expressing cells were mixed with control cells at the ratio of 25% or more. In the in vivo experiments, HRA transfectants were injected intraperitoneally. The number of intraperitoneal tumors of HRA/NK4 was much smaller than that of control. In mice injected with HRA/NK4, ascites formation was suppressed and the survival was significantly prolonged. These findings suggest that NK4-mediated gene therapy can improve the prognosis of ovarian cancer by suppressing peritoneal dissemination.

Animals↗

PubMed1986

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.

Amino Acids↗

PubMed2002

Microsatellite instability in cancer: what problems remain unanswered?

Analyses of microsatellite instability have been prevalent, particularly in the field of oncology. However, the literature on this subject is diverse. The discrepancies may derive from methodological problems in the conventional techniques used for analysis. Problems include low quantitativity in the detection systems, inaccurate migration in electrophoresis, and Taq polymerase-mediated modifications of polymerase chain reaction products. Indeed, use of a new fluorescent technique where these problems have been overcome has elucidated various intriguing and previously unrecognized aspects of microsatellite instability in human cancers. Patterns of microsatellite changes observed in various human cancers can be classified into 2 subtypes, those showing relatively small changes within 6 base pairs (type A) and those exhibiting drastic changes over 8 base pairs (type B). Although type A microsatellite instability has been connected to defective mismatch repair phenotype, the relationship between type B microsatellite instability and defective mismatch repair phenotype remains unclear. Nevertheless, as symbolized in cases of hereditary nonpolyposis colorectal cancer, connections between type B microsatellite instability and familial predisposition have been suggested in some cancers. The molecular background of type B microsatellite changes warrants particular attention.

Colorectal Neoplasms↗

PubMed2005

Sphere formation and expression of neural proteins by human corneal stromal cells in vitro.

PURPOSE: To demonstrate the presence of corneal stromal precursors that express neural markers in vitro. METHODS: To isolate sphere-forming cells, human corneal stromal cells were subjected to a reaggregation-free neurosphere assay in medium containing methylcellulose gel matrix. To promote differentiation, the isolated sphere colonies were plated in wells with medium containing fetal bovine serum. Expression of nestin, vimentin, keratocan, alpha-smooth muscle actin (alphaSMA), beta-III tubulin, neurofilament M (NFM), and glial fibrillary acidic protein (GFAP) was examined in the sphere colonies and their progeny (i.e., cells migrated from sphere colonies), by immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Human corneal stromal cells formed sphere colonies that had no self-renewal capability. The frequency of sphere-forming cells was 1.5% +/- 0.1% (range, 1.3%-1.6%). Most of the cells within these colonies expressed nestin and vimentin, whereas some expressed beta-III tubulin, NFM, GFAP, and alphaSMA by immunocytochemistry. Ninety-one percent and 89% of the progeny expressed vimentin and alphaSMA, respectively, whereas nestin was undetectable. beta-III tubulin-, NFM-, and GFAP-positive cells were detected in the progeny at the frequency of 7.2%, 0.9%, and 0.5%, respectively. Semiquantitative RT-PCR showed that nestin, NFM, GFAP, and keratocan gene expression was higher in the sphere colonies, whereas vimentin and alphaSMA expression increased in the progeny. CONCLUSIONS: Adult human corneal stroma contains precursor cells that express neural markers.

Adult↗

PubMedDate not supplied

Tuning of the selectivity in capillary electrophoresis by cyclodextrins illustrated by the separation of some structurally related phenothiazines.

Cyclodextrins were used to affect the selectivity of the capillary electrophoresis system in the separation of 10 widely used phenothiazines. It was shown that the addition of cyclodextrins substantially improved the selectivity. The effect of temperature and cyclodextrin concentration was studied on the resolution between the screened phenothiazines. The best results were obtained with 8 mM hydroxypropyl-beta-cyclodextrin at 15.5 degrees C. Under these conditions, a resolution of at least 1.5 between all phenothiazines could be obtained. In addition, the chiral separation of the enantiomers of trimeprazine could be accomplished. Structure separability relations between the phenothiazines showed that a change in the side chain at the R10 position had the greatest effect on the migration.

Cyclodextrins↗

PubMed1991

Alterations in proteoglycan synthesis common to healing wounds and tumors.

Wound healing and tumor stroma generation share several important properties, including hyperpermeable blood vessels, extravasation of fibrinogen, and extravascular clotting. In both, the deposits of fibrin gel serve initially as provisional stroma and later are replaced by granulation tissue. Proteoglycans (PG) are also important constituents of the extracellular matrix, but their composition and role in healing wounds and tumor stroma generation are poorly understood. The authors used immunohistochemical and biochemical methods to investigate the dermatan sulfate proteoglycan (DSPG) and chondroitin sulfate proteoglycan (CSPG) composition of healing skin wounds and solid tumors. By immunohistochemistry, the great majority of normal guinea pig and human dermis stained weakly for CSPG and strongly for decorin. In contrast, the granulation tissue of healing skin wounds and scars stained intensely for CSPG and weakly or not at all for decorin; however decorin staining was restored to normal intensity after digestion with chondroitin ABC lyase, suggesting that decorin antigenic sites had been masked by glycosaminoglycan (GAG) chains. Like wounds, the stroma of several carcinomas (line 1 guinea pig, human breast, colon, basal cell, and squamous) stained strongly for CSPG and weakly or not at all for decorin, but decorin staining developed after chondroitin ABC lyase digestion. Thus healing wounds and tumor stroma express a common pattern of altered PG staining, adding another to the properties these pathologic entities share. Proteoglycans extracted from healing wounds after in situ labelling with [35S] Na sulfate contained more CSPG than normal dermis with significantly longer GAG chains. Granulation tissue also synthesized more DSPG than normal skin, with greater heterogeneity and longer GAG chains. These alterations in PG synthesis correlate with the cell proliferation, migration, and collagen synthesis that accompany wound healing and may provide clues to the mechanisms responsible for both wound healing and tumor stroma generation.

Animals↗

PubMed1980

Trypsin activation of human factor XI.

Human factor XI circulates as a zymogen composed of two similar or identical chains of Mr = 80,000. Upon activation either by trypsin or by blood-clotting proteins involving clotting factors XII and high molecular weight kininogen, it undergoes proteolytic cleavage in which the Mr = 80,000 chain reportedly is cleaved to a heavy and light chain of Mr of about 48,000 and 33,000, respectively. In these studies, we have reinvestigated trypsin activation of factor XI and demonstrate that trypsin-activated factor XI contains three chains of apparent Mr = 46,000, 37,000, and 26,000. Kinetic studies lead to the conclusion that the parent chain of Mr = 80,000 is cleaved into chains of Mr = 46,000 and 37,000. This cleavage is followed by a second nondestructive cleavage, most probably of the chain of Mr = 46,000, to yield the third product which migrates as a band of Mr = 26,000.

Enzyme Activation↗

PubMed2003

[Effect of salvianolate on migration of human vascular endothelial cells].

OBJECTIVE: To study the effect of salvianolate on the migration of endothelial cells induced by monocytes. METHODS: Transwell-boydom system was used to test the migration of endothelial cells induced by monocytes. Enzyme-linked immunosorbent assay (ELISA) and RT-polymerase chain reaction (RT-PCR) method were used to determine the effect of salvianolate on the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and their mRNA of monocytes. RESULTS: The migration of endothelial cells induced by monocytes was facilitated by salvianolate, compared with the control. After the monocytes were treated by salvianolate for 24 hours, the expression of mRNA of VEGF and bFGF was significantly up-regulated, and the expression of VEGF and bFGF was increased. CONCLUSION: The facilitation of salvianolate on the migration of endothelial cells induced by monocytes was observed. Salvianolate stimulates the expression of VEGF and bFGF and their mRNA of monocytes, and salvianolate may have the role of inducing migration of endothelial cells by working on these two factors.

Cell Line↗