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At least 307 records · Page 17Linked to original sources
PubMed1997

A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent.

One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.

Animals↗
PubMed2006

Increased melanizing activity in Anopheles gambiae does not affect development of Plasmodium falciparum.

Serpins are central to the modulation of various innate immune responses in insects and are suspected to influence the outcome of malaria parasite infection in mosquito vectors. Three Anopheles gambiae serpins (SRPN1, -2, and -3) were tested for their ability to inhibit the prophenoloxidase cascade, a key regulatory process in the melanization response. Recombinant SRPN1 and -2 can bind and inhibit a heterologous phenoloxidase-activating protease and inhibit phenoloxidase activation in vitro. Using a reverse genetics approach, we studied the effect of SRPN2 on melanization in An. gambiae adult females in vivo. Depletion of SRPN2 from the mosquito hemolymph increases melanin deposition on foreign surfaces such as negatively charged Sephadex beads. As reported, the knockdown of SRPN2 adversely affects the ability of the rodent malaria parasite Plasmodium berghei to invade the midgut epithelium and develop into oocysts. Importantly, we tested whether the absence of SRPN2 from the hemolymph influences Plasmodium falciparum development. RNAi silencing of SRPN2 in an An. gambiae strain originally established from local populations in Yaoundé, Cameroon, did not influence the development of autochthonous field isolates of P. falciparum. This study suggests immune evasion strategies of the human malaria parasite and emphasizes the need to study mosquito innate immune responses toward the pathogens they transmit in natural vector-parasite combinations.

Animals↗
PubMed1997

Beta-amyloid peptides initiate the complement cascade without producing a comparable effect on the terminal pathway in vitro.

Activation of the classical complement cascade by beta-amyloid peptides has been hypothesized to underlie the neurodegeneration observed in Alzheimer's diseased brains. In this study, various lots of synthetic beta-amyloid peptides, A beta(1-40), A beta(1-42), and A beta(25-35), were tested for their ability to activate both early complement cascade events and formation of the membrane attack complex through terminal pathway activation. Unlike recent reports which did not assess activation of complement terminal pathway, we found that concentrations of beta-amyloid which activated early cascade events, to an extent comparable to aggregated IgG, failed to elicit formation of comparable levels of membrane attack complex.

Amyloid beta-Peptides↗
PubMed2006

Elastolysis induces collagenolysis in a gingival lamina propria model.

Elastin peptides were previously reported to increase MMP expression in several cell types. We found binding of these peptides to their receptors led to enhanced MMP-3 and MMP-1 expression, but not activation, in human gingival fibroblasts cultured on plastic dishes. We hypothesized that these peptides, in a more physiological environment, might additionally trigger an MMP-3/MMP-1 activation cascade, leading to matrix lysis, as occurs in periodontitis. To test this hypothesis, we used contracted and attached lattices as gingival lamina propria equivalents. In such 3D models, supplementation of elastin peptides and plasminogen triggered an MMP-3/MMP-1 activation cascade and significant down-regulation of TIMPs production, further leading to intense collagen degradation. We propose that elastolysis, as occurs in periodontitis, potentiates collagenolysis, thus promoting disease progression.

Adult↗
PubMedDate not supplied

Resolving liposomal inhibition of quantitative LAL methods.

Bacterial endotoxin is composed of Lipid A as well as variable polysaccharide regions, and is capable of eliciting a pyrogenic response in vivo (1). NeXstar has developed a quantitative LAL assay at maximum sensitivity to provide assurance that its products are well below the endotoxin pyrogenic threshold. This test uses rigorous treatment of 1.0 mL liposome samples with sodium dodecyl sulfate, an anionic surfactant, or CHAPS, a zwitterionic surfactant to disrupt the lipids present in the liposome sample including endotoxin. This liposome disruption allows potentially undetectable endotoxin to become available to the LAL cascade for quantification and overcomes inhibition of the LAL test by liposomes.

Endotoxins↗
PubMed1997

Intravenous morphine increases release of nitric oxide from spinal cord by an alpha-adrenergic and cholinergic mechanism.

Systemic opioids produce analgesia in part by activating bulbospinal noradrenergic pathways. Spinally released norepinephrine (NE) has been suggested to produce analgesia in part by stimulating alpha2-adrenoceptors on cholinergic spinal interneurons to release acetylcholine (ACh). We hypothesized that this spinally released ACh would stimulate synthesis of nitric oxide (NO), and that spinally released NO after intravenous (IV) opioid injection thus would depend on a cascade of noradrenergic and cholinergic receptor stimulation. To test these hypotheses, IV morphine was administered to anesthetized sheep, and neurotransmitters in dorsal horn interstitial fluid were measured by microdialysis. IV morphine increased NE and ACh in dorsal horn microdialysates, and these increases were inhibited by IV naloxone or cervical spinal cord transection. IV morphine also increased dorsal horn microdialysate concentrations of nitrite, a stable metabolite of NO. Increases in NE, ACh, and nitrite were antagonized by prior intrathecal injection of the alpha2-adrenergic antagonist idazoxan, the muscarinic antagonist atropine, or the NO synthase inhibitor N-methyl--arginine (NMLA). To examine the concentration-dependent effects of spinal adrenergic stimulation, isolated rat spinal cord tissue was perfused with the alpha2-adrenergic agonist clonidine. Clonidine increased nitrite in the spinal cord tissue perfusate, an effect blocked by coadministration of idazoxan, atropine, and NMLA. These data support a previously hypothesized cascade of spinally released NE and ACh after systemic opioid administration. These data also suggest that spinally released NO plays a role in the analgesic effects of systemic opioids. In addition, these data imply a positive feedback whereby spinally released nitric oxide increases NE release and that has not previously been described.

Acetylcholine↗
PubMed2000

Automatic classification of protein sequences into structure/function groups via parallel cascade identification: a feasibility study.

A recent paper introduced the approach of using nonlinear system identification as a means for automatically classifying protein sequences into their structure/function families. The particular technique utilized, known as parallel cascade identification (PCI), could train classifiers on a very limited set of exemplars from the protein families to be distinguished and still achieve impressively good two-way classifications. For the nonlinear system classifiers to have numerical inputs, each amino acid in the protein was mapped into a corresponding hydrophobicity value, and the resulting hydrophobicity profile was used in place of the primary amino acid sequence. While the ensuing classification accuracy was gratifying, the use of (Rose scale) hydrophobicity values had some disadvantages. These included representing multiple amino acids by the same value, weighting some amino acids more heavily than others, and covering a narrow numerical range, resulting in a poor input for system identification. This paper introduces binary and multilevel sequence codes to represent amino acids, for use in protein classification. The new binary and multilevel sequences, which are still able to encode information such as hydrophobicity, polarity, and charge, avoid the above disadvantages and increase classification accuracy. Indeed, over a much larger test set than in the original study, parallel cascade models using numerical profiles constructed with the new codes achieved slightly higher two-way classification rates than did hidden Markov models (HMMs) using the primary amino acid sequences, and combining PCI and HMM approaches increased accuracy.

Algorithms↗
PubMed1998

Synovium as a source of increased amino-terminal parathyroid hormone-related protein expression in rheumatoid arthritis. A possible role for locally produced parathyroid hormone-related protein in the pathogenesis of rheumatoid arthritis.

Proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 (IL-1), mediate the joint destruction that characterizes rheumatoid arthritis (RA). Previous studies have shown that parathyroid hormone-related protein (PTHrP) is a member of the cascade of proinflammatory cytokines induced in parenchymal organs during lethal endotoxemia. To test the hypothesis that NH2-terminal PTHrP, a potent bone resorbing agent, could also be a member of the synovial cascade of tissue-destructive cytokines whose expression is induced in RA, PTHrP expression was examined in synovium and synoviocytes obtained from patients with RA and osteoarthritis (OA). PTHrP production, as determined by measurement of immunoreactive PTHrP(1-86) in tissue explant supernatants, was increased 10-fold in RA versus OA synovial tissue. Synovial lining cells and fibroblast-like cells within the pannus expressed both PTHrP and the PTH/PTHrP receptor, findings that were confirmed by in vitro studies of cultured synoviocytes. TNF-alpha and IL-1beta stimulated PTHrP expression in synoviocytes, while dexamethasone and interferon-gamma, agents with some therapeutic efficacy in the treatment of RA, inhibited PTHrP release. Treatment of synoviocytes with PTHrP(1-34) stimulated IL-6 secretion. These results suggest that proinflammatory cytokine-stimulated production of NH2-terminal PTHrP by synovial tissue directly invading cartilage and bone in RA may mediate joint destruction through direct effects on cartilage or bone, or, indirectly, via the induction of mediators of bone resorption in the tumor-like synovium.

Arthritis, Rheumatoid↗
PubMed2000

Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae.

In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor --> G protein --> phospholipase C --> inositol trisphosphate --> intracellular Ca(2+) concentration ([Ca(2+)](i)) --> calmodulin --> myosin light chain kinase --> phosphorylated myosin --> force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca(2+)](i) concurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01-0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca(2+)](i) is still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca(2+)](i).

Animals↗
PubMed1985

Fluosol-DA causes pulmonary hypertension and increased lung lymph flow in unanesthetized sheep.

Test doses (0.5 ml) of the perfluorochemical blood substitute Fluosol-DA causes pulmonary hypertension in some patients. To investigate this phenomenon we infused Fluosol-DA into unanesthetized sheep with chronic vascular catheters on ten occasions. In six of these experiments lung lymph flow was measured. Since other fluorochemical blood substitutes alter the coagulation cascade we also sampled blood to perform coagulation screening tests and look for evidence of thrombin generation as assessed by the survival of 125I labelled sheep fibrinogen. Test doses (0.5 ml) of Fluosol-DA resulted in transient but marked pulmonary hypertension in nine of ten experiments (17 +/- 1.2 SE to 43 +/- 3.9 SE torr). Cardiac output decreased by an average of 30%, and lung lymph flow transiently increased without a change in the lymph to plasma protein concentration (L/P) ratio in five of six experiments. When 50 ml of Fluosol-DA was administered one hour later the pulmonary hypertension was more prolonged but less severe (18 +/- 1.0 SE to 34 +/- 3.2 SE torr). Lymph flow again increased without a change in the L/P ratio for protein. The activated partial thromboplastin and prothrombin times, and survival in plasma of 125I labelled sheep fibrinogen were unaffected by administration of Fluosol-DA. We conclude that Fluosol-DA causes pulmonary hypertension in sheep and increases lung lymph flow. This results from vasoconstriction of the pulmonary vessels and subsequent recruitment of the pulmonary microvasculature and not from thromboembolic phenomena.

Animals↗
PubMed2002

Postsynaptic kinase signaling underlies inhibitory synaptic plasticity in the lateral superior olive.

UNLABELLED: In the auditory system, inhibitory transmission from the medial nucleus of the trapezoid body (MNTB) to neurons of the lateral superior olivary nucleus (LSO) undergoes activity-dependent long-term depression, and may be associated with developmental elimination of these synapses [Sanes DH, Friauf E (2000). REVIEW: development and influence of inhibition in the laterial superior olivary nucleus. Hear Res 147:46-58]. Although GABA(B) receptor activation and postsynaptic free calcium are implicated in this depression, little is known about intracellular signaling mechanisms in this or other forms of inhibitory plasticity. In this study, we asked whether the calcium dependency of inhibitory depression was associated with the activation of calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and/or cAMP-dependent protein kinase A (PKA). Whole-cell voltage-clamp recordings were obtained from LSO neurons in a brain slice preparation, permitting for the selective pharmacologic manipulation of individual postsynaptic LSO neurons. Inclusion of a CaMKII antagonist (KN-62) in the internal pipet solution blocked inhibitory synaptic depression. A second CaMKII inhibitor (autocamtide peptide fragment) significantly decreased inhibitory depression. Inclusion of a specific antagonist of protein kinase C (PKC fragment 19-36) in the internal recording solution also blocked inhibitory depression. To test involvement of a cAMP-dependent intracellular cascade, two different manipulations were performed. Inclusion of PKA antagonists (Rp-cAMPS or a cAMP dependent protein kinase inhibitor peptide) prevented inhibitory depression. In contrast, when a nonhydrolyzable cAMP analog (Sp-cAMPS) was permitted to enter the postsynaptic cell, the MNTB-evoked IPSCs became depressed in the absence of low-frequency stimulation. Thus, three key postsynaptic kinases, CaMKII, PKC, and PKA, participate in the activity-dependent depression of inhibitory MNTB-LSO synapses during postnatal development.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2004

Cytokinin-induced hypocotyl elongation in light-grown Arabidopsis plants with inhibited ethylene action or indole-3-acetic acid transport.

Cytokinins inhibit hypocotyl elongation in darkness but have no obvious effect on hypocotyl length in the light. However, we found that cytokinins do promote hypocotyl elongation in the light when ethylene action is blocked. A 50% increase in Arabidopsis thaliana (L.) Heynh. hypocotyl length was observed in response to N6-benzyladenine (BA) treatment in the presence of Ag+. The level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid was strongly increased, indicating that ethylene biosynthesis was up-regulated by treatment with cytokinin. Furthermore, the effects of cytokinins on hypocotyl elongation were also tested using a series of mutants in the cascade of the ethylene-signal pathway. In the ethylene-insensitive mutants etr1-3 and ein2-1, cytokinin treatment resulted in hypocotyl lengths comparable to those of wild-type seedlings treated with both Ag+ and BA. A similar phenotypical response to cytokinin was observed when auxin transport was blocked by alpha-naphthylphthalamic acid (NPA). Applied cytokinin largely restored cell elongation in the basal and middle parts of the hypocotyls of NPA-treated seedlings and at the same time abolished the NPA-induced decrease in indole-3-acetic acid levels. Our data support the hypothesis that, in the light, cytokinins interact with the ethylene-signalling pathway and conditionally up-regulate ethylene and auxin synthesis.

Arabidopsis↗
PubMed2004

On-line estimation of biomass concentration using a neural network and information about metabolic state.

This paper deals with the design of a neural network-based biomass concentration estimation system. This system is enhanced by the incorporation of information about the actual metabolism of the microorganism cultivated, which is taken from an on-line knowledge-based system. Two different design approaches have been investigated using the fed-batch cultivation of baker's yeast as the model process. In the first, metabolic state (MS) data were passed as additional input to the neural network; in the second, these data were used to select a neural network suitable for the specific MS. Two neural network types--feed-forward (Levenberg-Marquardt) and cascade correlation--were applied to this system and tested, and the performances of these neural networks were compared.

Algorithms↗
PubMed1980

The efficiency of various humidifiers for respired gas.

Eight different breathing gas humidifiers were tested by humidity measurements using two independent methods under simulated clinical conditions. The comparison considering the influence of tidal volume and inspiratory/expiratory ratios revealed great differences between the individual devices depending on the kind of humidification and functional principles. Only four of the humidifiers tested: Ultrasonic Nebulizer NB 108, Hydrotrop 200, Bennett Cascade and Nicholas Humidifier, gave a constant performance of more than 30.75 g H2O/m3 absolute humidity or 70% relative humidity, related to 37 degrees C. However, humidification and heating of the inspired gas could be ensured only by the last three devices. The humidifiers Bird 500 cm3 Micronebulizer, Ultrasonic Nebulizer NB 100 and Draeger Compressor 660, were strongly dependent on the respiration parameters and exceeded the required minimum of 70% relative humidity only under certain conditions. The Engstroem ER-200 Vaporizer had a low performance and was not suitable as a breathing gas humidifier for longterm ventilation.

Humans↗
PubMed2002

THIS: a tuneable heterodyne infrared spectrometer.

With the Cologne Tuneable Heterodyne Infrared Spectrometer (THIS) we present a newly developed setup of a transportable heterodyne receiver. Competitiveness with regard to sensitivity, was reached for the first time with a semiconductor laser pumped system. Frequency tuneability of the local oscillator (LO) laser over a wide range of wavelengths is thus provided. This allows a variety of molecules, e.g. O3, NH3, CH4, N2O,.... in the earth's atmosphere, in planetary atmospheres or even in interstellar space to be observed with very high frequency resolution either from aboard the Stratospheric Observatory For Infrared Astronomy (SOFIA) or other ground based telescopes. Besides the good results with tuneable lead salt laser (TDL) operation there's room to improve: the power provided by such devices is not sufficient for a sensitivity close to the quantum limit. Therefore, first tests with recently developed high power quantum-cascade lasers (QCL) were carried out and further substantial improvement of the system noise temperature seems to be in reach.

Spectrophotometry, Infrared↗
PubMed2005

The antidepressant imipramine inhibits M current by activating a phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent pathway in rat sympathetic neurones.

Little is known about the intracellular actions of imipramine (IMI) in the regulation of ion channels. We tested the action of IMI on the intracellular cascade that regulates M current (I(M)) in superior cervical ganglion neurones (SCGs). Dialysis of the cells with GDPbetaS, a G protein signaling blocker, did not disrupt the inhibition of I(M). When we incubated the cells with the phospholipase C (PLC) inhibitor U73122, it prevented the I(M) inhibition by IMI. Also, when we dialyzed the cells with an intracellular Ca2+ chelator, it did not disrupt I(M) inhibition by IMI, as occurs in the M1 cascade. When we incubated the cells with the generic kinase inhibitor wortmannin, it prevented the recovery of I(M) from the inhibition by IMI. Also, when we applied phosphatidylinositol 4,5-bisphosphate (PIP2) intracellularly, it diminished the inhibition of I(M) by IMI. Our findings suggest that PLC is the target for IMI, that recovery of I(M) needs lipid phosphorylation for PIP2 resynthesis, and that IMI inhibits I(M) by activating a PLC-dependent pathway, likely by decreasing the concentration of PIP2.

Animals↗
PubMed2001

Distinct carbohydrate recognition domains of an invertebrate defense molecule recognize Gram-negative and Gram-positive bacteria.

Coelomic fluid of Eisenia foetida earthworms (Oligochaeta, Annelida) contains a 42-kDa defense molecule named CCF for coelomic cytolytic factor. By binding microbial antigens, namely the O-antigen of lipopolysaccharide (LPS), beta-1,3-glucans, or N,N'-diacetylchitobiose present, respectively, on Gram-negative bacteria or yeast cell walls, CCF triggers the prophenoloxidase activating pathway. We report that CCF recognizes lysozyme-predigested Gram-positive bacteria or the peptidoglycan constituent muramyl dipeptide as well as muramic acid. To identify the pattern recognition domains of CCF, deletion mutants were tested for their ability to reconstitute the prophenoloxidase cascade in E. foetida coelomic fluid depleted of endogenous CCF in the presence of LPS, beta-1,3-glucans, N,N'-diacetylchitobiose, and muramic acid. In addition, affinity chromatography of CCF peptides was performed on immobilized beta-1,3-glucans or N,N'-diacetylchitobiose. We found that the broad specificity of CCF for pathogen-associated molecular patterns results from the presence of two distinct pattern recognition domains. One domain, which shows homology with the polysaccharide and glucanase motifs of beta-1,3-glucanases and invertebrate defense molecules located in the central part of the CCF polypeptide chain, interacts with LPS and beta-1,3-glucans. The C-terminal tryptophan-rich domain mediates interactions of CCF with N,N'-diacetylchitobiose and muramic acid. These data provide evidence for the presence of spatially distinct carbohydrate recognition domains within this invertebrate defense molecule.

Amino Acid Sequence↗
PubMed2003

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone.

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.

Animals↗