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Results for “Extracellular Traps”

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PubMed1992

An in-vitro study of chloroquine transport in the rat submaxillary gland.

The uptake and efflux of chloroquine by the rat submaxillary gland in-vitro were studied under various incubation conditions. Variations in the extracellular pH significantly affected both the uptake and efflux of the drug. Increasing chloroquine concentration significantly decreased the uptake. Uptake was also decreased significantly (P less than 0.05) when compared with control conditions (pH 7.40, 37.5 degrees C, O2 aeration, 6 x 10(-6) M chloroquine) by the following experimental variations: aeration of the incubation medium with N2 instead of O2; decrease of bath temperature from 37.5 to 4 degrees C; addition of metabolic inhibitors, cyanide (10(-3) M), iodoacetic acid (10(-3) M) and o-nitrophenol (10(-3) M). Cimetidine (10(-3) M), a known organic cationic inhibitor, had no significant effect on chloroquine uptake when compared with control values. These results show that the uptake of chloroquine by the rat submaxillary gland in-vitro is concentration-dependent and this is indicative of possible saturable binding sites for the drug in the gland. These results suggest that the transfer of chloroquine across the submaxillary gland may be mediated by an active transport process. On the other hand, it is possible that the apparent active transport process implicated in this study could be a consequence of chloroquine ion trapping in the acidic interior of lysosomes.

Animals↗

PubMed2003

Regulation of interleukin-3-induced substrate phosphorylation and cell survival by SHP-2 (Src-homology protein tyrosine phosphatase 2).

The cytosolic SHP-2 (Src homology protein tyrosine phosphatase 2) has previously been implicated in IL-3 (interleukin-3) signalling [Bone, Dechert, Jirik, Schrader and Welham (1997) J. Biol. Chem. 272, 14470 -14476; Craddock and Welham (1997) J. Biol. Chem. 272, 29281-29289; Welham, Dechert, Leslie, Jirik and Schrader (1994) J. Biol. Chem. 269, 23764-23768; Qu, Nguyen, Chen and Feng (2001) Blood 97, 911-914]. To investigate the role of SHP-2 in IL-3 signalling in greater detail, we have inducibly expressed WT (wild-type) or two potentially substrate-trapping mutant forms of SHP-2, generated by mutation of Asp-425 to Ala (D425A) or Cyst-459 to Ser (C459S), in IL-3-dependent BaF/3 cells. Effects on IL-3-induced tyrosine phosphorylation, signal transduction and functional responses were examined. Expression of C459S SHP-2 protected the beta-chain of the murine IL-3R (IL-3 receptor), the adaptor protein Gab2 (Grb2-associated binder 2), and a cytosolic protein of 48 kDa from tyrosine dephosphorylation, consistent with them being bona fide substrates of SHP-2 in IL-3 signalling. The tyrosine phosphorylation of a 135 kDa transmembrane protein was also protected upon expression of C459S SHP-2. We have identified the inhibitory immunoreceptor PECAM-1 (platelet endothelial cell adhesion molecule-1)/CD31 (cluster determinant 31) as a component of this 135 kDa substrate and also show that IL-3 can induce tyrosine phosphorylation of PECAM-1. Expression of WT, C459S and D425A forms of SHP-2 had little effect on IL-3-driven proliferation or STAT5 (signal transduction and activators of transcription) phosphorylation or activation of protein kinase B. However, expression of WT SHP-2 increased ERK (extracellular-signal-regulated kinase) activation. Interestingly, expression of C459S SHP-2 decreased ERK activation at later times after IL-3 stimulation, but potentiated IL-3-induced activation of Jun N-terminal kinases. In addition, expression of C459S SHP-2 decreased cell survival in suboptimal IL-3 and upon IL-3 withdrawal. These findings indicate that SHP-2 plays an important role in mediating the anti-apoptotic effect of IL-3 and raises the possibility that PECAM-1 participates in the modulation of cytokine-induced signals.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics.

1. Structurally distinct superoxide dismutase (SOD) mimetics were examined for their ability to protect nitric oxide (NO) from destruction by oxidant stress in rabbit aorta. 2. These were the spin traps, PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide), tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) and tiron (4,5-dihydroxy-1,3-benzene-disulphonic acid), the metal salts, CuSO4 and MnCl2, and the metal-based agents CuDIPS (Cu (II)-[diisopropylsalicylate]2) and MnTMPyP (Mn (III) tetrakis [1-methyl-4-pyridyl]porphyrin). 3. Oxidant stress was generated in isolated aortic rings by inactivating endogenous Cu/Zn SOD with diethyldithiocarbamate (DETCA; 60 min) either alone at 3 mM or at 0.3 mM in combination with superoxide generation using xanthine oxidase (XO; 4.8 mu ml(-1)) and hypoxanthine (HX; 0.1 mM). 4. Acetylcholine (ACh)-induced relaxation was inhibited by DETCA (3 mM, 60 min) and was not restored by exogenous SOD (250 u ml(-1)), suggesting the oxidant stress was intracellular. MnTMPyP (600 microM and 1 mM) and MnCl2 (100 microM) were the only agents to reverse the blockade of ACh-induced relaxation. 5. Addition of XO/HX to DETCA (0.3 mM)-treated tissues powerfully impaired ACh-induced relaxation and exogenous SOD (250 u ml(-1)) fully reversed the blockade, suggesting the oxidant stress was extracellular. CuDIPS (0.1-3 microM), CuSO4 (0.3-3 microM), MnCl2 (1-100 microM) and MnTMPyP (100-600 microM) also reversed blockade powerfully, tempol (30 microM-1 mM) and tiron (0.3-10 mM) reversed blockade weakly and PTIYO (10-300 microM) enhanced the blockade. 6. Thus, MnTMPyP was the only SOD mimetic to restore NO-dependent relaxation in conditions of both extracellular and intracellular oxidant stress. This agent may, therefore, provide a lead in the development of SOD mimetics for the treatment of pathologies associated with oxidant stress.

Acetylcholine↗

PubMed1990

Glutathione conjugation protects some, but not all, cell lines against DNA binding of benzo[alpha]pyrene metabolites.

Three major types of cell lines were distinguished according to their capacity for glutathione (GSH) conjugation of extracellularly generated benzo[alpha]pyrene (BaP) metabolites, and the level of DNA binding of such metabolites. (i) Cells, e.g. HepG2, which conjugate BaP metabolites only very poorly with GSH and are highly susceptible to DNA binding. The number of DNA adducts in these cells (approximately 10 pmol/mg DNA) is taken to be the maximum DNA binding in 'unprotected' cells. (ii) Cells, e.g. V79 and NCI-H322, which efficiently conjugate BaP metabolites with GSH but, like HepG2 cells, are 'unprotected' as indicted by maximum DNA binding. (iii) Cells, e.g. 2sFou and H4IIEC3/G-, which are positive for GSH conjugation and exhibit only very little DNA binding. When GSH conjugation in these apparently 'protected' cells is suppressed by depletion of GSH, the level of DNA binding increases to that found in 'unprotected' lines. GSH depletion does not substantially affect DNA binding in 'unprotected' cells. The results show that, although GSH conjugation is capable of suppressing DNA binding of reactive BaP metabolites in some cell types, it fails to protect against DNA binding in others. It is possible that the reactive species are compartmentalized and certain cell types are protected, because they are able to specifically trap those BaP metabolites which bind to DNA.

Animals↗

PubMed1990

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion.

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.

CD18 Antigens↗

PubMed2004

Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism.

Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A "hit-and-run" type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in G2/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in Ser25 phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-1. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ("the Golgi blockade hypothesis").

Animals↗

PubMed1987

Intracellular free calcium concentration in rat anterior pituitary cells as indicated by fura-2: effect of arginine-vasopressin.

Arginine-vasopressin (AVP) stimulates adrenocorticotropin and beta-endorphin release from corticotrophs of the anterior pituitary gland through mechanisms which are not initiated by an elevation of the cellular levels of adenosine-3',5'-cyclic-monophosphate. In the present study the effect of AVP on the cytoplasmic concentrations of free calcium ions in rat anterior pituitary cells was examined. Cytosolic free Ca2+ concentrations were monitored directly using the new, intracellularly trapped fluorescent indicator fura-2. In cells incubated in medium containing 1.3 mmol/l Ca2+, AVP (100 nmol/l) caused an immediate elevation of the cytoplasmic Ca2+ concentration by about 50 nmol/l (P less than 0.001). The intracellular Ca2+ levels remained elevated during the observation period of 2-3 min. This effect of AVP was blocked by a specific vasopressin antagonist. By contrast, the glucocorticoid dexamethasone did not affect the AVP-induced elevation of cytosolic Ca2+ concentration. When the cells were incubated in Ca2+-free medium (Ca2+ omitted, EGTA 2 mmol/l), the AVP-induced as well as the K+ depolarization-induced increase in free cytoplasmic Ca2+ were abolished, whereas the ionophore ionomycin evoked a rapid transient elevation of free Ca2+. The increase in cytoplasmic Ca2+ concentration induced by AVP was preserved in medium containing the calcium channel blockers Mg2+ (Mg2+ 31.2 mmol/l; Ca2+ 1.3 mmol/l) or nifedipine (1 mumol/l). The potassium-evoked calcium signal was blocked by Mg2+ (31.2 mmol/l). We conclude that vasopressin induces a rapid rise in the cytoplasmic concentration of free calcium ions in corticotrophs. Vasopressin may mobilize calcium through mechanisms that neither are glucocorticoid-sensitive nor involve the influx of extracellular calcium through voltage-dependent calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing.

A model is presented outlining the molecular and cellular events that occur during the early stages of the wound healing process. The underlying theme is that there is a specific binding interaction between fibrin, the major clot protein, and hyaluronic acid (HA), a constituent of the wound extracellular matrix. This binding interaction, which could also be stabilized by other cross-linking components, provides the driving force to organize a three-dimensional HA matrix attached to and interdigitated with the initial fibrin matrix. The HA-fibrin matrix plays a major role in the subsequent tissue reconstruction processes. We suggest that HA and fibrin have both structural and regulatory functions at different times during the wound healing process. The concentration of HA in blood and in the initial clot is very low. This is consistent with the proposed interaction between HA and fibrin(ogen), which could interfere with either fibrinogen activation or fibrin assembly and cross-linking. We propose that an activator (e.g. derived from a plasma precursor, platelets or surrounding cells) is produced during the clotting reaction and then stimulates one or more blood cell types to synthesize and secrete HA into the fibrin matrix of the clot. We predict that HA controls the stability of the matrix by regulating the degradation of fibrin. The new HA-fibrin matrix increases or stabilizes the volume and porosity of the clot and then serves as a physical support, a scaffold through which cells trapped in the clot or cells infiltrating from the peripheral edge of the wound can migrate. The HA-fibrin matrix also actively stimulates or induces cell motility and activates and regulates many functions of blood cells, which are involved in the inflammatory response, including phagocytosis and chemotaxis. The secondary HA-fibrin matrix itself is then modified as cells continue to migrate into the wound, secreting hyaluronidase and plasminogen activator to degrade the HA and fibrin. At the same time these cells secrete collagen and glycosaminoglycans to make a more differentiated matrix. The degradation products derived from both fibrin and HA are, in turn, important regulatory molecules which control cellular functions involved in the inflammatory response and new blood vessel formation in the healing wound. The proposed model generates a number of testable experimental predictions.

Blood Coagulation↗

PubMed2000

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.

Acid Phosphatase↗

PubMed1990

Reperfusion damage: free radicals mediate delayed membrane changes rather than early ventricular arrhythmias.

STUDY OBJECTIVE - The aim of the study was to reassess the role of reactive oxygen species in causing reperfusion arrhythmias, which they might do either by directly generating free oxygen radicals or by using scavengers of free oxygen radicals. DESIGN - Ventricular arrhythmias were studied in isolated rat hearts (n = 8-15 per experiment) subjected to regional ischaemia and treated with various free radical scavengers and spin trap agents. Reoxygenation automaticity was similarly studied in isolated guinea pig papillary muscles (n = 6-13 per experiment). MEASUREMENTS and RESULTS - In isolated rat hearts early reperfusion ventricular arrhythmias were unaltered by superoxide dismutase (1 X 10(5) IU.litre-1), catalase (1 X 10(6) IU.litre-1), N-tert-butyl-alpha-phenylnitrone (30 mumols.litre-1), 5,5-dimethyl-1-pyrroline-N-oxide (1 mmol.litre-1), or the combination of superoxide dismutase 1 X 10(5) IU.litre-1, catalase 1 X 10(6) IU.litre-1, and mannitol 10 mol.litre-1, or by the generation of the free radical .OH (Fe:ADP plus dihydroxyfumerate). In the isolated reoxygenated guinea pig papillary muscle, the incidence of reoxygenation automaticity was significantly reduced by verapamil 5 mumols.litre-1 but not by the following free oxygen radical scavengers: reduced glutathione (0.5 mmol.litre-1), N-acetyl cysteine (1 mmol.litre-1), the combination of superoxide dismutase (3 X 10(4) IU.litre-1) and catalase (5 X 10(3) IU.litre-1), or by pretreatment with allopurinol (30 mg.kg-1). Generating systems of .O2- or .OH induced relatively slow electrophysiological changes, including a decreased action potential duration. Reperfusion ventricular fibrillation in the rat heart was increased by increasing the extracellular calcium concentration from 1.25 to 1.9 or 2.5 mmol.litre-1, or by prolongation of the ischaemic time. CONCLUSIONS - Because of (a) the lack of an arrhythmogenic effect of free radical generating systems or of scavengers of free radicals, (b) the calcium sensitivity of reperfusion arrhythmias, and (c) the relatively slow time course of electrophysiological changes induced by free radical generating systems, we propose that free radicals are unlikely to be the prime cause of early ventricular arrhythmias in the systems that we tested. The mechanism of such arrhythmias is more likely to be a calcium sensitive process. The relatively slow electrophysiological changes mediated by free radicals suggest that these agents can cause delayed membrane change.

Action Potentials↗

PubMed1990

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles.

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36-48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell-cell contacts involved in a) the formation of the thyroid follicular lumen and b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC-Dextran and Cascade Blue conjugated to bovine serum albumin: CB-BSA). LY microinjected into IL of 2-9-day-old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 microns. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC-Dextran or CB-BSA microinjected into IL behaved as LY; the probes were restrained into the lumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15-30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2(+)-dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FITC-Dextran or CB-BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell-to-cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell-to-cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.

Acridine Orange↗

PubMed1991

Chimeric antithrombin peptide. Characterization of an Arg-Gly-Asp (RGD)- and hirudin carboxyl terminus-containing synthetic peptides.

We investigated the properties of an artificial chimeric peptide that contains an Arg-Gly-Asp (RGD)-tripeptide, the versatile cell recognition signal of extracellular matrix protein components, coupled to a carboxyl-terminal fragment of the highly specific alpha-thrombin inhibitor, hirudin (residues 53-64): WGRGDSANGDFEEIPEEYL (RGD-hirudin53-64). Hirudin53-64 and RGD-hirudin53-64 inhibited the fibrinogen clotting activity of alpha-thrombin and prolonged the activated partial thromboplastin time of human plasma. In addition, both peptides afforded total protection to thrombin from trypsionolysis. Neither hirudin53-64 nor RGD-hirudin53-64 dramatically interfered with the thrombin-antithrombin inhibition reaction either in the absence or presence of added heparin. alpha-Thrombin-induced platelet aggregation was effectively inhibited by hirudin53-64 and RGD-hirudin53-64. Unlike hirudin53-64, RGD-hirudin53-64 in solution inhibited integrin-mediated endothelial cell and fibroblast cell attachment to polystyrene wells in the presence of fetal bovine serum. Collectively, our results demonstrate that RGD-hirudin53-64 has anticoagulant/antiplatelet aggregation activity attributable to its hirudin sequence and integrin-directed cell attachment activity due to its RGD site. Our results suggest that this chimeric motif may serve as a prototype for a new class of anticoagulants where an integrin-specific sequence "targets" the peptide to a cell (ultimately through the platelet integrin alpha IIb beta 3) trapped amid a thrombus with ensuing proteinase inhibition.

Amino Acid Sequence↗

PubMed1999

Protein conformational changes in the bacteriorhodopsin photocycle.

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation. Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.

Bacteriorhodopsins↗

PubMed1997

Risk of neural tube defect-affected pregnancy is associated with a block in maternal one-carbon metabolism at the level of N-5-methyltetrahydrofolate:homocysteine methyltransferase.

The disposition of whole blood mono-to hexaglutamyl methylfolate and plasma homocysteine (HCY) was used to evaluate potential lesion sites in one-carbon metabolism which could be responsible for neural tube defect(NTD)-affected pregnancies. An isocratic high-performance liquid chromatographic system (HPLC) with photodiode array detection was used to quantify and speciate whole-blood methylfolate into mono-, di-, tri-, tetra-, penta-, and hexaglutamate forms. This technique was also used with off-line radioassay to identify nonmethyl whole-blood folates. Isocratic HPLC with fluorescence detection was used to quantify SBDF derivatized homocysteine in plasma. The study investigated blood from 11 women who had experienced a previous NTD-affected pregnancy and 11 controls of similar age and social class. No subjects were pregnant. HCY levels were significantly higher in NTD subjects (P = 0.0486, 95% CI-2.799,0.001 using the Mann-Whitney test), as was the ratio of known intracellular (tri-to hexaglutamyl) methylfolate compared to extracellular (mono- and diglutamyl) methylfolate (P = 0.0062 95% CI-0.543, 3.862 using the Mann-Whitney test). Vitamin B12, red cell folate, circulating total methylfolate, and circulating mono-to hexaglutamyl methylfolates showed no difference between population groups. The disposition between individual and cumulative glutamate chain lengths of methylfolate showed significant trends which differed between population groups: (i) total blood methylfolate (Glu1-6) appears to be utilized by N-5-methyltetrahydrofolate:homocysteine methyltransferase (MS) in control blood but not NTD blood, where it appears to accumulate following a 45-min incubation; (ii) whole-blood hexaglutamyl methylfolate (5CH3-H4PteGlus) becomes a larger proportion of the total blood methylfolate in NTD than in control populations; and (iii) the intermediate glutamate chains of methylfolate (Glu1-5) remain relatively constant as 5CH3-H4PteGlu6 accumulates in NTD but appear to increase linearly with 5CH3-H4PteGlu6 in controls. The significant elevation of HCY in the NTD population is associated with the increasing proportion of 5CH3-H4PteGlu6 relative to the total methylfolate, since, when corrected for HCY level, the proportion of 5CH3-H4PteGlus to total methylfolate is similar in NTD and control populations. These trends are consistent with a defect at the level of vitamin B12 dependent MS which "traps" folate at the 5CH3-H4PteGlus level.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed2000

Structural dynamics and function of early embryonic coats.

The extracellular embryonic coats (embryo/blastocyst coverings) that surround early mammalian embryos are most often still referred to as zona pellucida. Accumulating evidence from a number of earlier and recent studies clearly indicates that this is an oversimplification which cannot be defended anymore, at least in many species. Structural modifications of the coats occur during cleavage and blastocyst stages; these are most obvious in a number of species with the central type of implantation and, related to this, a high degree of blastocyst expansion, notably the rabbit and the horse. In this contribution, formation and transformation of the various layers of coats (zona pellucida, mucoprotein layer, neozona and gloiolemma in the rabbit, capsule in the horse) will be reviewed, as will be comparable structures seen, e.g., in the fur seal (subzonal layer) and the baboon. These phenomena will be discussed in the context of structurally more subtle changes found in other species, including those with small blastocysts and other types of implantation, in particular with biochemical modifications which may be physiologically quite important. The molecular mechanisms of deposition of coats and their transformation and shedding/dissolution will be briefly addressed. The possible functional significance of the coats and their transformation will be discussed (mechanical, morphogenetic and immunological role, molecular transport control, blastocyst positioning, implantation, trap and reservoir function for signalling molecules).

Animals↗

PubMed1995

Pathogenesis of atherosclerosis.

The earliest lesion in the development of an atherosclerotic plaque is the fatty streak. This chronic inflammatory reaction results from a sequence of events that begins with the trapping of low density lipoprotein (LDL) in the subendothelial space of the artery wall. The trapped LDL is seeded with oxidative species released by the overlying endothelium, and lipid oxidation is initiated within the LDL particle. Some of the lipids that result lead to the activation of NFkB-like transcription factors that cause the expression of genes whose protein products mediate monocyte binding, monocyte chemotaxis into the subendothelial space, and conversion into macrophages. At least 1 major gene modulates the oxidation of LDL lipids and/or the biologic response to these lipids. The inverse relation between high density lipoprotein (HDL) and atherosclerotic events may in part be due to enzymes associated with HDL that destroy the biologically active lipids generated in LDL.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2001

ATP modulates Na+ channel gating and induces a non-selective cation current in a neuronal hippocampal cell line.

Extracellular ATP evoked two excitatory responses in hippocampal neuroblastoma cells (HN2). The first, an opening of a receptor-operated non-selective cation channel and the second was a leftward shift in Na+ channel activation. Both ATP (5-1000 microM) and 2',3'-(4-benzoyl)-benzoyl-ATP (Bb-ATP, 50 microM) activated a non-selective cation current reversing near 0 mV and shifted the Na+ activation and inactivation curves to the left. Based on a comparison of a series of agonists and antagonists, the inward current appeared to be partially mediated by activation of a P2X7 receptor, although hybrid channels cannot be ruled out. The shift in Na+ channel gating could be separated from the opening of the cation channel, as application of the P2Y antagonist Reactive Blue-2 and GTP shifted the Na+ current activation to the left but failed to elicit the inward cation current. Both responses to ATP and Bb-ATP were insensitive to block by the P2X antagonist suramin (300 microM) but were prevented by incubation in oxidized ATP (200 microM); a putative P2X7 receptor antagonist. Prior screening of the surface negative charge of the membrane with a high concentration of divalent cations prevented both responses. We suggest that ATP4- activates a P2X receptor and becomes trapped on a site, on or near the Na+ channel. Activation of the P2X receptor leads to the opening of a non-specific cation channel, while the binding of ATP4- leads to a modified charge sensed by the Na+ channel, similar to what occurs in the presence of charged amphiphiles as well as a number of beta-scorpion toxins.

Adenosine Triphosphate↗

PubMed1998

Intercellular material in the basal and lateral folds of parotid serous cells in four species of bats.

BACKGROUND: Basal folds are slender plications at the basal surface of acinar cells in the salivary glands of many mammalian species. These largely organelle-free folds increase the surface area of the basal plasmalemma manyfold and are unquestionably involved in the translocation of organic and inorganic molecules and water into the acinar cells. METHODS: Specimens of salivary glands were obtained from over 230 species of live-trapped bats from major areas of the globe. Tissues for electron microscopy were fixed and processed by conventional means. RESULTS: A number of the bat species examined had dense material in the intercellular spaces between basal and lateral folds of serous cells in the parotid gland. This intercellular material was particularly prominent in three species of New World bats, viz., Pteronotus parnellii, P quadridens, and Phyllostomus latifolius, and in one species of Old World bats, Chalinolobus argentatus. This dense material, which has a farinaceous texture, appears not to pass through tight junctions, so it is excluded from the lumina of intercellular canaliculi and acini. The dense material originates in the acinar cells--it is carried to the membranes of the folds via coated vesicles, which empty their dense content by exocytosis into the intercellular space. Similar dense material is present in the intercellular spaces of the basal labyrinth of striated ducts in the two species of Pteronotus. The manner in which this material accumulates in the striated duct is unclear. CONCLUSIONS: Although the function of the intracellular dense material is undetermined, it appears to be placed strategically to influence molecular traffic into acinar cells or to modulate the paracellular pathway. From a comparative evolutionary perspective, we hypothesize that, in bats, the combination of basal folds and extracellular densities is associated with insectivory. Similar morphologies appear to be lacking in frugivorous or nectarivorous species.

Animals↗