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At least 253 records · Page 14Linked to original sources
PubMed1995

On-pathway versus off-pathway folding intermediates.

Rapidly formed molten globule intermediates accumulate at the start of the folding reactions of several small proteins. Opinion is sharply divided as to whether they are on-pathway or off-pathway intermediates. I discuss recent experiments aimed at resolving this issue. Specific points include whether a 'rollover' in the plot of folding rate versus denaturant concentration implies that a folding intermediate is or is not on-pathway; whether the failure to observe folding intermediates for some small proteins implies a different folding mechanism or only that the intermediates are less stable; possible interpretation of 'fast-track' folding of hen lysozyme; and the significance of recent results in the search for unfolding intermediates.

Journal Article↗
PubMed1997

A novel degradation pathway of tissue factor pathway inhibitor: incorporation into fibrin clot and degradation by thrombin.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2, and K3) that regulates the initial reactions of the extrinsic blood coagulation pathway through K1 and K2. In the present study, the effect of thrombin on TFPI in a purified system was first examined using recombinant TFPI from Chinese hamster ovary (CHO) cells. TFPI was inactivated by thrombin with cleavage of three peptide bonds, Lys 254-Thr 255 in the C-terminal basic region, Arg 107-Gly 108 (reactive site toward factor Xa in K2), and Lys 86-Thr 87 between K1 and K2. Then, degradation of radiolabeled TFPI by thrombin was examined in two systems: (1) mixed with plasma and then tissue factor (TF) and calcium ion, and (2) mixed with fibrinogen and then thrombin. TFPI degradation was detected in serum from normal plasma and more extensively from anti-thrombin (AT)-depleted plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Significant radioactivity was found in the clot after coagulation of the plasma, which decreased after 20 hours' incubation. These changes were more prominent in AT-depleted plasma than in normal plasma. When TFPI lacking the C-terminal basic region was used instead of full-length TFPI, most of the radioactivity was found in serum rather than in fibrin clots. Incorporation of TFPI into the fibrin clot was prevented by a synthetic C-terminal peptide of TFPI. Similar results were obtained after mixing radiolabeled TFPI with fibrinogen and then thrombin in the presence of calcium ion or EDTA. These results demonstrate a novel degradation pathway of TFPI, ie, incorporation into fibrin via the C-terminal basic region and degradation by thrombin (possibly fibrin-bound thrombin).

Animals↗
PubMed1982

A one-step procedure for preparation of classical pathway (C1q) and alternative pathway (factor D) depleted human serum.

A simple method for preparation of a serum depleted in both C1q and Factor D is described. The hemolytic activities of both pathways are completely abolished and can be fully restored using the respective purified complement components. Furthermore, this serum is useful for studying cell systems since blocking either complement pathway does not require chelating agents.

Binding, Competitive↗
PubMed2003

On the site of C4 deposition upon complement activation via the mannan-binding lectin pathway or the classical pathway.

The mannan-binding lectin (MBL) pathway and the classical pathway of complement activation are initiated by the binding of the recognition structure of the initiator complexes, MBL and C1q, respectively, to their ligands, i.e. carbohydrate structures or immune complexes. Proenzymes associated with MBL or C1q are then activated and generate C3 convertase through the activation of C4 and C2. The cleavage product of C4, C4b, attaches covalently to nearby hydroxyl or amino groups. The current picture is that C2 must then attach to C4b before being cleaved by the same associated proteases into the enzymatically active fragment, C2b. This suggests a stringent requirement for the deposition of C4b very close to the initiator complex, or indeed onto the initiator complex. We examined the possibility of C4b being bound to the initiator complex by a solid-phase assay, allowing for the selective elution of the initiator complexes, followed by quantification of the C4b being eluted and the C4b remaining on the solid phase. Also, we estimated the generation of complexes between the released initiator complex and C4b. More than 99% of deposited C4b was bound directly to the solid phase rather than to the initiator complex. Our approach cannot answer the question of the whereabouts of the C2 when it is cleaved.

Binding Sites↗
PubMed1979

Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation.

Living Schistosoma mansoni schistosomula incubated with normal chicken, guinea pig, human, and monkey sera were killed after 4 hr contact at 37 degrees C. The following data indicate that this action is dependent on the activation of the alternative complement pathway (AP): a) the inactivity of RB, RD, and zymosan-treated serum against schistosomula; b) the partial activity of RD restored in FD; c) the full effect of the C4-deficient guinea pig, C2-deficient human, and the agammaglobulinemic human sera; d) the consumption of both the AP and FB after the incubation of NHS with schistosomula; e) the detection of C3d breakdown product during the contact of the C2-deficient human serum with these young parasites. Killing by serum was decreased as the immature schistosomes developed and was completely absent against 4-day-old lung schistosomula (LS). In other experiments, it was demonstrated that schistosomula, in the presence of IgG, were able to initiate complement activation also through the classical pathway (CP). However, the CP does not appear to play a role in the schistosomulicidal activity of complement. The in vivo relevance of these observations is considered.

Animals↗
PubMed1986

Alternative complement pathway activation by C4b deposited during classical pathway activation.

Sheep erythrocytes (E) sensitized with anti-E antibody (A) were reacted with guinea pig C1 (C1gp) and human C4 (C4hu) or guinea pig C4 (C4gp) to prepare EAC1, 4b. Treatment of the EAC1, 4b with a buffer containing EDTA removes C1rgp and C1sgp, resulting in the formation of EAC4b. EAC4b prepared in this way were found to be lysed by human or guinea pig serum in a gelatin Veronal-buffered saline containing 2 mM MgCl2 and 8 mM EGTA (Mg-EGTA-GVB). In the hemolytic sensitivity of EAC4bhu, essentially no difference was noted whether IgG or IgM antibodies were used for preparation of EAC4bhu. The extent of the hemolysis of EAC4bhu was dependent on the dose of C4bhu. Because EAC4bhu were lysed even by C2-deficient human serum, C3 convertase of the classical complement pathway would not be involved in the hemolysis of EAC4bhu. Furthermore, the reactivity of EAC4bhu with serum in Mg-EGTA-GVB remained even after treatment of the intermediate cells with 1 mM PMSF, indicating that any remaining C1gp was not responsible for the hemolysis. Therefore, the hemolysis of EAC4b by sera in Mg-EGTA-GVB was considered to be mediated via activation of the alternative complement pathway (ACP). Pretreatment of EAC4bhu with anti-C4hu antibody or C4-binding protein suppressed the hemolysis of EAC4bhu via the ACP activation. Furthermore, EAC4bhu were more sensitive to hemolysis by the reaction with a mixture of C3, B, D, and H followed by rat serum in EDTA-GVB than EAC1qgp were. These results indicate that C4b molecules on the cell membrane participate in the activation of ACP.

Animals↗
PubMed1984

The influence of classical pathway components during alternative pathway--modulated immune complex aggregation: the role of C1 INH.

Alternative pathway (AP)-triggered reactions as well as classical pathway (CP)-mediated ones, were investigated turbidimetrically and/or immune electrophoretically, either in the presence or in the absence of in situ-generated immune complexes (ICs; tetanus toxoid/human anti-tetanus toxoid-IgG; ICs of equivalence) during the early stages of reaction. Monospecific Fab'- or Fab-fragments (rabbit) were used to block the complement function in normal human serum (NHS). C1q, functionally available following the addition of ethylene-glycol-bis-(beta-aminoethyl ether), N,N'-tetraacetic acid to NHS (EGTA-NHS), was found to increase the IC aggregation, thereby producing a biological surface upon which AP-dependent proteins were deposited. The functional inhibition of C1INH caused a C1s-mediated C3 conversion irrespective of the fact whether C1s was incorporated within macromolecular C1 (NHS) or dissociated from it (EGTA-NHS), thus, in the latter case inhibiting the AP-dependent portion of turbidity. It seemed probable that C3 conversion was effected by a fluid-phase CP C3 convertase. This process, normally counteracted by C1INH, worked more efficiently in EGTA-NHS than in NHS, indicating that the C1s-mediated reactions, initiated by presently unknown mechanisms, were less extensively regulated outside of the Ca2+-dependent C1 complex. The study demonstrates that in EGTA-NHS, too, where AP-triggered reactions have usually been investigated, sections of CP activation may play an important role, especially in situations where the function of C1INH is restricted.

Antigen-Antibody Complex↗
PubMed1988

Dorsal raphe stimulation modulates nociceptive responses in thalamic parafascicular neurons via an ascending pathway: further studies on ascending pain modulation pathways.

A study on the nociceptive responses of single cells within the nucleus parafascicularis (PF) thalami of the rat was undertaken to clarify the reported observations of a pain suppression pathway to this nucleus from the dorsal raphe (DR) nucleus. Two types of nociceptive neuron were identified in the PF which were classified as 'nociceptive-on' and 'nociceptive-off' neurons, respectively. DR stimulation exhibits a simple monophasic 'dose-dependent' relationship between the degree of the inhibition elicited and the stimulation intensity used on the 'nociceptive-off' cells. In contrast, biphasic effects following DR stimulation on the 'nociceptive-on' cells was obtained, with low intensities eliciting suppression while high intensities excited the cells. These effects of low intensity DR stimulation upon the responses of the 'nociceptive-on' cells were diminished but not prevented by transection of the well-known bulbospinal inhibitory fibers descending in the dorsal half of the spinal cord, while the effects of DR stimulation upon the 'nociceptive-off' cells remain unchanged following spinal transection. Thus, our results show that DR stimulation modulates the nociceptive responsiveness of the PF by way of supraspinal pathways in addition to the previously described descending paths.

Animals↗
PubMed2002

Pathways for emotion: interactions of prefrontal and anterior temporal pathways in the amygdala of the rhesus monkey.

The amygdala has been implicated in processing information about the emotional significance of the environment and in the expression of emotions, through robust pathways with prefrontal, anterior temporal areas, and central autonomic structures. We investigated the anatomic organization and intersection of these pathways in the amygdala in rhesus monkeys with the aid of bidirectional, retrograde and anterograde tracers. Connections of the amygdala with orbitofrontal and medial prefrontal areas were robust and bidirectional, whereas connections with lateral prefrontal areas were sparse, unidirectional and ascending. Orbitofrontal axons terminated densely in a narrow band around the borders of the magnocellular basolateral nucleus, surrounded by projection neurons along a continuum through the nuclei of the basal complex. In contrast, the input and output zones of medial prefrontal areas were intermingled in the amygdala. Moreover, medial prefrontal axonal terminations were expansive, spreading into the parvicellular basolateral nucleus, which is robustly connected with hypothalamic autonomic structures, suggesting that they may influence the expressive emotional system of the amygdala. On the other hand, orbitofrontal axons heavily targeted the intercalated masses, which issue inhibitory projections to the central nucleus, at least in rats and cats. The central nucleus, in turn, issues a significant inhibitory projection to hypothalamic and brainstem autonomic structures. This evidence suggests that orbitofrontal areas exercise control on the internal processing of the amygdala. In addition, the results provided direct evidence that the connections of anterior temporal visual and auditory association cortices occupy overlapping territories with the orbitofrontal cortices particularly in the posterior half of the amygdala, and specifically within the intermediate sector of the basolateral nucleus and in the magnocellular part of the basomedial nucleus (also known as accessory basal), suggesting a closely linked triadic network. This intricate network may be recruited in cognitive tasks that are inextricably linked with emotional associations.

Amygdala↗
PubMed1999

The pain-related negative difference potential: a direct measure of central pain pathway activity or of interactions between the innocuous somatosensory and pain pathways?

Negative difference potential (NDP) is a sural nerve-evoked scalp potential derived by subtracting potentials elicited at the pain threshold level from those elicited at supra-pain threshold levels. Our recent work examined the possibility that the NDP reflects a pain-related inhibition of neurons in the innocuous somatosensory pathways. Although failing to find any evidence for this inhibition, these studies do present the possibility that the NDP reflects an attention- and/or task-related decrease in the innocuous somatosensory activity that is elicited by the noxious electrical stimulus. To test this hypothesis, 35 healthy subjects were given three attention/task relevance conditions presented in counterbalanced order: rate the subjective magnitude of the painful aspects of the noxious electrical stimulus; rate the subjective magnitude of the non-painful aspects of the noxious electrical stimulus; and, ignore the stimulus. Neither changes in attention nor the task relevance of the non-painful aspects of the stimulus had any effect on NDP amplitude. These data demonstrate that the NDP does not reflect an attention- or task-related modulation of innocuous somatosensory activity. Rather, our evidence to date suggests that the NDP is generated by activity in the central pain pathways.

Adult↗
PubMed1985

Nociceptive pathways: anatomy and physiology of nociceptive ascending pathways.

In primates, the principal nociceptive pathways ascend in the anterolateral quadrant of the spinal cord. Among these, the spinothalamic tract (s.t.t.) is the best studied. Cells in Rexed's laminae I and V project to the ventro-posterolateral (v.p.l.) thalamic nucleus. Other cells in the same and deeper laminae terminate in the intralaminar complex. Spinothalamic tract cells may be nociceptive-specific or multireceptive. Those ending in v.p.l. have restricted, contralateral receptive fields, whereas those projecting to the intralaminar region often have large, bilateral receptive fields. Spinoreticular tract (s.r.t.) cells are concentrated in laminae VII and VIII and may be nociceptive. It is proposed that the s.t.t. contributes to sensory-discriminative processing of pain and that the s.t.t. and s.r.t. play a role in the motivational-affective components of pain. Alternative nociceptive pathways are the spinocervical and postsynaptic dorsal column tracts.

Afferent Pathways↗
PubMed1993

L-arginine exerts a dual role in nociceptive processing in the brain: involvement of the kyotorphin-Met-enkephalin pathway and NO-cyclic GMP pathway.

1. Intracerebroventricular (i.c.v.) administration of L-arginine (L-Arg), at 10-100 micrograms per mouse, produced antinociception in mice, as assessed by the tail flick test; this antinociception was reversed by pretreatment (s.c.) with naltrindole (NTI), a delta-selective opioid antagonist, and by co-administered L-leucyl-L-arginine (Leu-Arg), a kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist. 2. L-NG-nitroarginine methyl ester (L-NAME), a NO synthase inhibitor, but not D-NG-nitroarginine methyl ester, given i.c.v. at 3-10 micrograms per mouse, exhibited antinociceptive activity that was resistant to naloxone (s.c.), NTI (s.c.) and Leu-Arg (i.c.v.). 3. The L-NAME (i.c.v.)-induced antinociception was not reversed by L-Arg (i.c.v.), which was antinociceptive by itself, but was abolished by combined injection of L-Arg plus Leu-Arg (i.c.v.) or by L-Arg (i.c.v.) after NTI (s.c.). 4. Methylene blue (MB), a soluble guanylate cyclase inhibitor, at 0.1-1 microgram per mouse, produced antinociception by i.c.v. administration. The antinociception induced by MB (i.c.v.) or L-NAME (i.c.v.) was reversed by co-administered dibutyryl cyclic GMP. 5. These findings suggest that L-Arg plays a dual role in nociceptive processing in the brain, being antinociceptive via the kyotorphin-Met-enkephalin pathway and nociceptive via the NO-cyclic GMP pathway.

Analgesics↗
PubMed1996

Imaging evaluation of the optic nerve and visual pathway including cranial nerves affecting the visual pathway.

The radiologic investigation of the optic pathways has an integral part in the diagnostic evaluation of diverse lesions, such as inflammatory disease, vascular disorders, and benign and malignant tumors that afflict the optic pathways. These radiologic methods consist principally of computed tomography and magnetic resonance imaging, and, in vascular lesions, magnetic resonance angiography and conventional angiography.

Cranial Nerve Neoplasms↗
PubMed1998

Aspirin increases the bleeding side effects in essential thrombocythemia independent of the cyclooxygenase pathway: role of the lipoxygenase pathway.

Acetylsalicylic acid (ASA) is currently recommended as an antithrombotic for patients with essential thrombocythemia (ET) who are at an increased risk of thrombotic events. However, ASA is also associated with an increased risk of bleeding in these patients as compared to the risk of bleeding in other patients treated with ASA. Recent data suggest that while ASA inhibits platelet thromboxane A2 (TxA2) synthesis in all individuals, ASA has little effect or inhibits the lipoxygenase pathway (i.e., 12-hydroxyeicosatetranoic acid or 12-HETE synthesis) in some individuals, and enhances 12-HETE synthesis in others. These differential effects are associated with a pronounced prolongation of the bleeding time vs. no prolongation of the bleeding time, respectively, i.e., in ASA responders and ASA nonresponders, respectively. To determine if the increased risk of ASA-induced bleeding seen in ET patients is associated with an effect on 12-HETE synthesis, we compared the relative effects of ASA on the bleeding time, platelet TxA2 and 12-HETE synthesis, and platelet aggregation and adhesion in ET patients and healthy volunteers. ASA (300 mg, taken orally) prolonged the bleeding time in 82% of the ET patients but only 27% of the healthy volunteers although platelet TxA2 synthesis and ADP- and collagen-induced aggregation were inhibited significantly in both groups. In contrast, platelet 12-HETE synthesis was unchanged and platelet adhesion was decreased in those patients and volunteers whose bleeding times were prolonged by ASA, whereas platelet 12-HETE synthesis was increased significantly and platelet adhesion was unaffected in those patients and volunteers whose bleeding times were not prolonged, and in some cases shortened by ASA. These results confirm previous data that demonstrate that ASA has different effects on platelet 12-HETE synthesis and platelet adhesion in different individuals, i.e., inhibitory or no effect in ASA responders (in whom ASA prolonged bleeding) vs. enhancing effects in ASA nonresponders (in whom ASA did not prolong bleeding). These results also indicate that there is a greater percentage of ASA responders in patients with ET than that seen in the general population, a difference that is associated with an effect of ASA on the lipoxygenase pathway. This may explain the increased bleeding side effects seen in the ET patient population.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed2000

Transforming growth factor-alpha-mediated growth pathways in human gastro-intestinal cell lines in relation to the gastrin autocrine pathway.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) increase transcription of the gastrin gene, and the gastrin peptide may be phosphorylated by EGF-stimulated tyrosine kinase. Our aims were to compare EGF/TGF-alpha interactions in 2 human gastro-intestinal cell lines: MGLVA1, with a strong gastrin autocrine pathway, and C170HM2, with a weak pathway. Both cell lines expressed the TGF-alpha gene. MGLVA1 expressed TGF-alpha protein as determined by immuno-cytochemistry, which was absent in C170HM2. Both cell lines expressed the same level of EGF receptors, as assessed by flow cytometry; however, MGLVA1 did not have enhanced in vitro proliferation in response to EGF or TGF-alpha, unlike C170HM2. The basal growth of MGLVA1 was inhibited by anti-sera against TGF-alpha, the EGF receptor and G17. C170HM2 was not inhibited by any of the anti-sera. Neutralisation of TGF-alpha resulted in undetectable cell-associated progastrin levels in MGLVA1 (untreated had 391.7 fmol/5 x 10(6) cells). The progastrin level of C170HM2 remained unaffected. Tyrosine kinase activity, as assessed by phosphopeptide concentration, of unstimulated MGLVA1 was 2.6 times higher than that of C170HM2 in the cell membrane fraction (0.097 compared to 0.037 microg/mg protein, p < 0.001) and 4.8 times higher in the cytosolic fraction (0.269 compared to 0.056 microg/mg protein, p < 0.05). Following treatment with EGF, the phosphopeptide concentration increased in both the membrane and cytosolic fractions of both cell lines. Tyrphostin B42, which inhibits autophosphorylation of the EGF receptor, inhibited the basal growth of MGLVA1 (IC(50) 1.3 microM) and C170HM2 (9.5 microM, p < 0.05 from MGLVA1). Herbimycin, which inhibits pp60(c-src) kinase, reduced the basal growth of MGLVA1 (0.67 microM) but not C170HM2. Immunofluorescence studies confirmed the presence of tyrosine-phosphorylated proteins and pp60(c-src) within the cytoplasm of unstimulated MGLVA1 cells. There was no specific immunofluorescence for either parameter in C170HM2 cells until after treatment with EGF.

Blotting, Southern↗
PubMed1993

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.

Amino Acid Sequence↗
PubMed1994

Detection of metabolites of the Entner-Doudoroff pathway by HPLC with pulsed amperometry: application to assays for pathway enzymes.

Three major metabolites in the Entner-Doudoroff pathway, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, and pyruvate can be detected and quantified by HPLC with pulsed amperometric detection. Resolution is achieved by ion-exchange chromatography at alkaline pH with isocratic elution in 5 to 10 min. Detection limits are in the subnanomolar range, and detector response is linear over 3-4 orders of magnitude. This method can be employed for the assay of the enzymes of the pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14), eliminating the need for coupling enzymes as in the previously employed spectrophotometric assays. The lag in pyruvate production seen in the coupled enzyme spectrophotometric assay for 6-phosphogluconate dehydratase is absent in the HPLC/pulsed amperometric detection assay. This lag represents an artifact of a slow tautomerism of 2-keto-3-deoxy-6-phosphogluconate which must precede its utilization by the coupling enzyme, 2-keto-3-deoxy-6-phosphogluconate aldolase. Kinetic data on the approach to equilibrium of 2-keto-3-deoxy-6-phosphogluconate aldolase-catalyzed interconversion of 2-keto-3-deoxy-6-phosphogluconate, pyruvate, and glyceral-dehyde-3-phosphate can be also accurately quantified by HPLC with pulsed amperometric detection.

Aldehyde-Lyases↗
PubMed1996

Embryonic development of Schwann cells: multiple roles for neuregulins along the pathway.

During the past few years important advances have been realized in the understanding of the molecular and cellular mechanisms operating in the differentiation of Schwann cells from neural crest cells. In particular the development of in vitro culture systems has allowed the analysis of commitment to the Schwann cell lineage, identification of an intermediate between the neural crest cell and the Schwann cell, and initial analysis of the factors controlling determination and differentiation processes. These studies point in particular to major roles for neuregulins which appear to be involved in the control of cell survival, proliferation, and differentiation, as well as of gene expression at different levels of the pathway.

Animals↗