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Results for “Extracellular Traps”

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PubMed2002

VEGF-Trap: a VEGF blocker with potent antitumor effects.

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.

3T3 Cells↗

PubMed1999

Presence of oxidized low density lipoprotein in nonrheumatic stenotic aortic valves.

The aim of the present study was to analyze if LDL particles trapped in stenotic aortic valve tissue undergo oxidative modification. Degenerative aortic stenosis affects >3% of the population >75 years of age in the Western world. Recent studies have revealed the presence of a chronic inflammatory process similar to what has been described in other degenerative diseases such as atherosclerosis. However, the underlying disease mechanisms of degenerative aortic stenosis still remain largely unknown. Six tricuspid stenotic valves, obtained at valve replacement, were compared with 3 control valves collected from hearts taken out during transplantation. The stenotic valves and the control valves were examined by immunohistochemistry, using antibodies against apoB, 4-hydroxynonenal-modified LDL, leukocytes, and HLA-DR. All valves were also stained with oil red O for neutral lipids. Extracellular neutral lipids were found in all stenotic valves, extending from the bases along the fibrosa layer. This lipid colocalized with apoB- and 4-hydroxynonenal-modified LDL immunoreactivity. 4-Hydroxynonenal-modified LDLs were present around calcium deposits, subendothelially, and in the deeper layer of the fibrosa. There was also a colocalization with macrophages, T lymphocytes, and HLA-DR expression. Control valves had a thin area of neutral lipid accumulation, a small amount of apoB, but no signs of inflammation. A distinct colocalization between oxidized LDLs, T-lymphocyte accumulation, and calcium deposits suggests that oxidized lipids may play a role in the disease process.

Adult↗

PubMed2003

The free radical spin-trap alpha-PBN attenuates periinfarct depolarizations following permanent middle cerebral artery occlusion in rats without reducing infarct volume.

The effect of the free radical spin-trap alpha-phenyl-butyl-tert-nitrone (alpha-PBN) in permanent focal cerebral ischemia in rats was examined in two series of experiments. In the first, rats were subjected to permanent occlusion of the middle cerebral artery (MCAO) and treated 1 h after occlusion with a single dose of alpha-PBN (100 mg/kg) or saline. Body temperature was measured and controlled for the first 24 h to obtain identical temperature curves in the two groups. Cortical infarct volumes were determined on histological sections 7 days later. alpha-PBN did not significantly reduce infarct volume (control: 28.3+/-16.3 mm3 vs. alpha-PBN 23.7+/-7.4 mm3). In the second series of experiments, periinfarct depolarizations (PIDs) were recorded with an extracellular DC electrode at two locations in the ischemic penumbra for the initial 3 h following MCAO. alpha-PBN (100 mg/kg, single dose in conjunction with occlusion) significantly reduced the total number (median value of 3 PIDs in the control groups vs. 1 PID in alpha-PBN groups, p<0.001) and total duration of the PIDs (median value 662 s in the control groups vs. 162 s in the alpha-PBN groups, p<0.006). In spite of this, cortical infarct volumes determined 7 days later in the same rats were not smaller in alpha-PBN-treated rats. The study thus demonstrates that attenuation of PIDs does not always lead to smaller infarcts if permanent arterial occlusion is followed by long survival time and does not support the hypothesis that PIDs per se are critical determinants of infarct size in this situation.

Animals↗

PubMed2000

Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat.

Parasynaptic communication, also termed volume transmission, has been suggested as an important means to mediate information transfer within the central nervous system. The purpose of the present study was to visualize by autoradiography the available channels for fluid movement within the extracellular space following injection of the inert extracellular marker [14C]inulin into the lateral ventricle or cisterna magna. Bolus injections of 5 microl of 1 microCi of [14C]inulin were made in awake rats via chronically implanted cannulae. After survival times ranging from 5 min to 4 h, brains were processed for in vivo autoradiography. At 5 min the tracer distributed throughout the ventricles, subarachnoid spaces and cisterns "downstream" of the injection sites. Penetration into the brain from these sites was complex with preferential entry along the ventral side of the brain, especially into the hypothalamus and brainstem. By 4 h virtually the entire brain was labeled irrespective of the site of tracer application. Sustained tracer entry from subarachnoid spaces suggests that some areas act as depots to trap circulating material. This mechanism may contribute to the pattern of deep penetration at later time-points. The spatial and temporal characteristics of fluid movement throughout the brain are instructive in the interpretation of many experimental procedures involving injection of molecules into the cerebrospinal fluid.

Animals↗

PubMed1999

Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules.

Platelet activation leads to secretion of granule contents and to the formation of microvesicles by shedding of membranes from the cell surface. Recently, we have described small internal vesicles in multivesicular bodies (MVBs) and alpha-granules, and suggested that these vesicles are secreted during platelet activation, analogous to the secretion of vesicles termed exosomes by other cell types. In the present study we report that two different types of membrane vesicles are released after stimulation of platelets with thrombin receptor agonist peptide SFLLRN (TRAP) or alpha-thrombin: microvesicles of 100 nm to 1 microm, and exosomes measuring 40 to 100 nm in diameter, similar in size as the internal vesicles in MVBs and alpha-granules. Microvesicles could be detected by flow cytometry but not the exosomes, probably because of the small size of the latter. Western blot analysis showed that isolated exosomes were selectively enriched in the tetraspan protein CD63. Whole-mount immuno-electron microscopy (IEM) confirmed this observation. Membrane proteins such as the integrin chains alpha(IIb)-beta(3) and beta(1), GPIbalpha, and P-selectin were predominantly present on the microvesicles. IEM of platelet aggregates showed CD63(+) internal vesicles in fusion profiles of MVBs, and in the extracellular space between platelet extensions. Annexin-V binding was mainly restricted to the microvesicles and to a low extent to exosomes. Binding of factor X and prothrombin was observed to the microvesicles but not to exosomes. These observations and the selective presence of CD63 suggest that released platelet exosomes may have an extracellular function other than the procoagulant activity, attributed to platelet microvesicles.

Blood Platelets↗

PubMed1986

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells.

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.

Acyclovir↗

PubMed2005

Treatment with extracellular HSP70/HSC70 protein can reduce polyglutamine toxicity and aggregation.

The accumulation of insoluble protein aggregates is a feature of neurodegenerative disease. Overexpression of Heat Shock Protein 70 (HSP70) can protect cells with protein aggregates from apoptosis. Another trait of HSP70 is its ability to cross the plasma membrane. Therefore, we purified a preparation of HSP70/HSC70 from bovine muscle and used it in a model of Huntington's disease. Human neuroblastoma SK-N-SH cells were transfected with huntington exon 1 with short (25) or long (103) CAG trinucleotide repeats coupled to green flourescent protein (GFP). Cells transfected with the long polyCAG repeat had insoluble protein aggregates and died through apoptosis. Biotinylated HSP70/HSC70 incorporated into the culture medium appeared inside the cells within 3-6 h of incubation. This incorporation correlated with a reduction in apoptotic cells by 40-50%. Confocal microscopy revealed that labelled internalized HSP70/HSC70 co-localized with the polyglutamine inclusions. The measurement of the number and size of inclusions showed that HSP70/HSC70 was able to reduce both these parameters. A filter trap assay and immunoblotting demonstrated that the introduction of HSP70/HSC70 also decreased protein aggregation. Together with earlier data on the effects of exogenously administered HSP70/HSC70 on cultured cells and on animals, these data show that preparations based on HSP70 may have some potential as therapies for a variety of neurodegenerative pathologies.

Animals↗

PubMed2004

A novel aminophospholipid transporter exclusively expressed in spermatozoa is required for membrane lipid asymmetry and normal fertilization.

Through the use of a functionally unbiased signal peptide trap screen, we have discovered an ATP-dependent aminophospholipid transporter that is exclusively expressed in the acrosomal region of spermatozoa; it is about 62% similar to the flippase, FIC1. We disrupted the transporter gene and found that the size of litters from male null mice was slightly smaller than found with wild-type males. Sperm morphology and motility were the same between null and wild-type littermates, but agents (merocyanine and annexin) that measure phospholipid packing or phosphatidylserine (PS) in the outer membrane leaflet showed that PS already existed in the outer leaflet of null spermatozoa before sperm capacitation. Fertilization rates were normal when null spermatozoa were added to zona pellucida-free eggs, but in the presence of the extracellular matrix, fewer transporter(-/-) spermatozoa bound tightly or penetrated the zona pellucida (ZP), and fewer underwent acrosome reactions. In vitro fertilization was compromised, especially at early time points or at low sperm concentrations after mixing null spermatozoa and eggs. Thus, a new aminophospholipid transporter expressed exclusively in spermatozoa is critical for normal phospholipid distribution in the bilayer, and for normal binding, penetration, and signaling by the zona pellucida.

Amino Acid Sequence↗

PubMed2006

Structural changes in the L photointermediate of bacteriorhodopsin.

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.

Amino Acid Sequence↗

PubMed2000

Metabolic depletion of ATP by fructose inversely controls CD95- and tumor necrosis factor receptor 1-mediated hepatic apoptosis.

Hepatocyte apoptosis is crucial in several forms of liver disease. Here, we examined in different models of murine liver injury whether and how metabolically induced alterations of hepatocyte ATP levels control receptor-mediated apoptosis. ATP was depleted either in primary hepatocytes or in vivo by various phosphate-trapping carbohydrates such as fructose. After the activation of the tumor necrosis factor (TNF) receptor or CD95, the extent of hepatocyte apoptosis and liver damage was quantified. TNF-induced cell death was completely blocked in ATP-depleted hepatocyte cultures, whereas apoptosis mediated by CD95 was enhanced. Similarly, acute TNF-induced liver injury in mice was entirely inhibited by ATP depletion with ketohexoses, whereas CD95-mediated hepatotoxicity was enhanced. ATP depletion prevented mitochondrial cytochrome c release, loss of mitochondrial membrane potential, activation of type II caspases, DNA fragmentation, and cell lysis after exposure to TNF. The extent of apoptosis inhibition correlated with the severity of ATP depletion, and TNF-induced apoptosis was restored when ATP was repleted by increasing the extracellular phosphate concentration. Our study demonstrates that TNF-induced hepatic apoptosis can be selectively and reversibly blocked upstream of mitochondrial dysfunction by ketohexose-mediated ATP depletion.

Adenosine Triphosphate↗

PubMed1997

Different effects of thrombin receptor activation on endothelium and smooth muscle cells of human coronary bypass vessels. Implications for venous bypass graft failure.

BACKGROUND: Thrombin is implicated in coronary bypass graft disease; it cleaves its receptor's extracellular N-terminal domain and unmasks a new N-terminus as a tethered ligand. We studied the effects of thrombin receptor activation in human internal mammary artery (IMA) and saphenous vein (SV). METHODS AND RESULTS: To study the effects of thrombin receptor activation on vasomotion, isolated blood vessels were suspended for isometric tension recording, and the effects on cell proliferation were studied in cultured smooth muscle cells (SMCs) of IMA and SV. Thrombin receptor expression in IMA and SV was analyzed by reverse transcription polymerase chain reaction and immunohistology. Receptor function was studied by analyzing the activation of mitogen-activated protein kinase (p42MAPK). In IMA thrombin evoked endothelium-dependent relaxations (65 +/- 5%) that were mimicked by thrombin receptor agonist peptide (TRAP) and reduced by the thrombin inhibitors recombinant (r-) hirudin and D-Phe-Pro-Arg-chloromethyl ketone (PPACK) (P < .05). In SV thrombin caused contractions (36 +/- 5% of 100 mmol/L KCl) that were inhibited by r-hirudin or PPACK (P < .05) but not mimicked by TRAP. In SMCs thrombin induced more pronounced [3H]thymidine incorporation (inhibited by r-hirudin or PPACK) in SV than IMA (P < .05), but activation of p42MAPK was similar in both vessels. TRAP induced weaker activation of p42MAPK than thrombin and did not stimulate [3H]thymidine incorporation in SMCs of SV or IMA. Immunohistology and RT-PCR demonstrated that the endothelium and SMCs of IMA and SV express thrombin receptor. CONCLUSIONS: Functional thrombin receptors are present on endothelium and SMCs of IMA and SV. Endothelial thrombin receptors mediate relaxation in IMA but not SV. Thrombin causes much more pronounced contraction and proliferation in SMCs of SV than IMA independent of tethered receptors, suggesting other thrombin receptors exist. These differences of thrombin receptor activation in IMA and SV may be important in the development of and therapy for graft disease.

Amino Acid Chloromethyl Ketones↗

PubMed1998

The pH dependent amelogenin solubility and its biological significance.

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habit of enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins in various pH (4.0-9.0) solutions with an ionic strength (IS) of 0.15 M using the Micro BCA protein assay at 25 degrees C or 37 degrees C. The solubility of the recombinant amelogenin rM179 was lowest (0.7 mg/ml) close to its isoelectric point and it increased below and above this point. The solubility of the recombinant amelogenin rM166 remained almost the same (1-2 mg/ml) as the pH rose from 6.0 to 9.0 and it increased as the solution became more acidic. Synthetic "tyrosine-rich amelogenin polypeptide" (TRAP) was extremely insoluble (<0.2 mg/ml) in the pH range studied while synthetic "leucine-rich amelogenin polypeptide" (LRAP) was readily soluble (>3.3 mg/ml). The native porcine amelogenin with apparent molecular weight 25 kDa shared similar solubility behavior to rM179. The porcine 23 kDa amelogenin was only sparingly soluble (0.3-0.8 mg/ml) over a wide range of pH. Interestingly, the porcine 20 kDa amelogenin was remarkably soluble in the pH range of 4.0 to 6.0 (approximately 12 mg/ml), but the solubility dropped strikingly to only approximately 0.2 mg/ml at pH larger than approximately 7.0. The strong dependence of amelogenin solubility on solution pH may be involved in the regulation of aggregation, enzymatic degradation and the binding properties of amelogenins, thus playing an important role in enamel biomineralization.

Amelogenin↗

PubMed1990

Ultrastructural localization of a tartrate-resistant acid ATPase in bone.

Osteoclasts are effector cells in bone breakdown, and the active bone resorption is confined to the ruffled border zone of these cells. An acid milieu is maintained in this zone which is probably a prerequisite for bone resorption. Tartrate-resistant acid phosphatase (TRAP) activity has been recognized as a characteristic property of osteoclasts and in several studies proposed as a cytochemical marker of osteoclasts. We have previously isolated and characterized a tartrate-resistant and iron-activated acid ATPase (TrATPase) from rat bone, the enzyme being a member of the TRAP family. In the present study the ultrastructural localization of this enzyme was delineated by employing immunogold technique on low temperature-embedded maxillar rat bone. Intensive immunolabeling was seen on the bone surfaces facing the ruffled border zone while lower amounts of marker were seen in adjacent bone areas, that is, on the bone surfaces facing the clear zone and deeper-into the bone. Within the osteoclasts gold markers were observed mainly in vesicular structures interpreted as lysosomes. Immunolabeling was also observed in the recently described endocytic cells located near osteoblasts and osteoclasts. Also in these cells, the marker was confined to lysosomelike structures. The amount of label in bone facing osteoblasts was low, as was the amount within osteoblasts. Our observation of extracellular localization, in particular accumulation of TrATPase in bone matrix facing the ruffled border area of the osteoclasts, favors the view that the enzyme is exported to areas of active bone resorption, thereby indicating a potential role for the enzyme in this process.

Adenosine Triphosphatases↗

PubMed2002

Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.

ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.

ATP Binding Cassette Transporter 1↗

PubMed2003

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide.

A fragment of the amyloid beta protein, betaA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that betaA(25-35) stimulated formation of ROS in concentration and time dependent manner. The inverted peptide, betaA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by betaA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to betaA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide (O2*-)- and hydroxyl (*OH)-radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of *OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of *OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to betaA(25-35). The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that betaA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA2. Production of O2*- can lead to HOCl and further formation of *OH, which both have a cytotxic potential.

Adult↗

PubMed1994

Cell fatty acid composition affects free radical formation during lipid peroxidation.

Lipid-derived free radicals generated from intact human U937 monocytes exposed to iron-induced oxidative stress were detected by electron paramagnetic resonance (EPR) with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Lipid radical formation was enhanced when the cells were enriched with n-3 or n-6 polyunsaturated fatty acids. Computer simulation indicated that at least two POBN spin adducts were formed, having spectral characteristics consistent with carbon-centered radicals (aN = 15.9 G and aH = 2.6 G; aN = 15.1 G and aH = 2.8 G). These alkyl radicals are probably formed by beta-scission of alkoxyl radicals. POBN spin adduct formation correlated with ethane generation. Addition of ascorbate to the assay medium greatly increased the radical signal intensity. Although radical generation was cell dependent and POBN spin adducts were observed in cell homogenates, the adducts formed by the intact cells were detected only in the extracellular medium. These findings indicate that the extent of lipid radical formation in response to oxidative stress can be influenced by changes in the polyunsaturated fatty acid composition of the cell lipids and suggest the possibility that carbon-centered lipi radicals may interact with extracellular structures.

Ascorbic Acid↗

PubMed1994

Mass-spectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus.

The amelogenins of the extracellular matrix of developing dental enamel, comprise a family of tissue-specific proteins which are postulated to play a central role in the biomineralization of dental enamel [1]. The primary structures of amelogenins derived from cow, pig, human, mouse and rat have now been elucidated by the interpretation of cDNA sequences or by direct amino acid sequence determinations [2-6] demonstrating a high degree of sequence homology between species [1]. However, the nature of post-translational modification of these proteins is less clear. In particular, early reports of amelogenin phosphorylation [7-8] have proved to be difficult to confirm by direct chemical analyses [1]. Using mass spectrographic analysis, we recently [9], reported that the lower molecular weight (5-7 kDa) bovine and porcine amelogenin polypeptides (TRAP and LRAP) contained a single phospho-serine residue at position 16Ser and, since these polypeptides are derived by proteolytic processing from the higher molecular weight "parent" amelogenins (18-25 kDa), we concluded that these precursor molecules must also be phosphorylated, as has previously been suggested [10]. In contrast to these observations, an extensive amino acid sequencing study of porcine amelogenins has recently reported no evidence for such phosphorylation [1]. We now report that a new analysis of the major porcine ("20K") amelogenin provides positive evidence for porcine amelogenin phosphorylation.

Amelogenin↗

PubMed2000

Differential sensitivity to hydroxyl radicals of pre- and postjunctional neurovascular transmission in the isolated canine mesenteric vein.

In some pathophysiological conditions, the first target of reactive oxygen intermediates is the vascular system. Superoxide anions, when generated in the vascular circulation, may then escape into the extracellular space via an anion channel and, following dismutation to hydrogen peroxide (H(2)O(2)), form hydroxyl radicals (HO(*)). In an attempt to understand the role of HO(*) in the regulation of transmission at the sympathetic neurovascular junction, the effect of HO(*) at nerve terminals was examined by measuring the amount of noradrenaline (NA) released from isolated, spirally cut, superfused canine mesenteric vein during basal and electrical stimulation (ES; 5Hz, 2ms, 9V); tension development evoked by ES was also recorded simultaneously. HO(*) was generated from Fenton's reagent (1. 5x10(-4)M H(2)O(2) plus 10(-4)M FeSO(4)); generation of HO(*) from H(2)O(2)/FeSO(4) in the superfusate was monitored by electron spin resonance spectroscopy using the spin-trap 5, 5-dimethyl-1-pyrroline-N-oxide throughout the experimental time course. Exposure to HO(*) of the helical strips produced an irreversible decrease in tension development evoked by ES with no effect on NA release, suggesting that the observed effect is elicited postjunctionally. The susceptibility of the processes of NA-mediated contraction to HO(*) may differ greatly from that of the NA release mechanism at the prejunctional site. Exposure of the strip preparation to HO(*) leads to a substantial stimulation of basal release of NA without affecting ES-evoked NA release, possibly due to enhanced non-exocytotic Ca(2+)-independent release elicited by HO(*). A direct demonstration of this concept was obtained by showing a significant increase in the basal response of NA release in Ca(2+)-free solution. The major conclusion of the present study is that HO(*) can damage NA-mediated contraction of the vascular preparations at the postjunctional site, and may selectively induce a non-exocytotic release of NA from the prejunctional site of sympathetic neurotransmission.

Animals↗