Addition Of C2 Research Articles

Decanoic Acid and Not Octanoic Acid Stimulates Fatty Acid Synthesis in U87MG Glioblastoma Cells: A Metabolomics Study.

Medium-chain fatty acids (MCFA) are dietary components with a chain length ranging from 6 to 12 carbon atoms. MCFA can cross the blood-brain barrier and in the brain can be oxidized through mitochondrial β-oxidation. As components of ketogenic diets, MCFA have demonstrated beneficial effects on different brain diseases, such as traumatic brain injury, Alzheimer’s disease, drug-resistant epilepsy, diabetes, and cancer. Despite the interest in MCFA effects, not much information is available about MCFA metabolism in the brain. In this study, with a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach, coupled with multivariate data analyses, we followed the metabolic changes of U87MG glioblastoma cells after the addition of octanoic (C8), or decanoic (C10) acids for 24 h. Our analysis highlighted significant differences in the metabolism of U87MG cells after the addition of C8 or C10 and identified several metabolites whose amount changed between the two groups of treated cells. Overall, metabolic pathway analyses suggested the citric acid cycle, Warburg effect, glutamine/glutamate metabolism, and ketone body metabolism as pathways influenced by C8 or C10 addition to U87MG cells. Our data demonstrated that, while C8 affected mitochondrial metabolism resulting in increased ketone body production, C10 mainly influenced cytosolic pathways by stimulating fatty acid synthesis. Moreover, glutamine might be the main substrate to support fatty acids synthesis in C10-treated cells. In conclusion, we identified a metabolic signature associated with C8 or C10 addition to U87MG cells that can be used to decipher metabolic responses of glioblastoma cells to MCFA treatment.

Effect of Aromatic Petroleum Resin on Damping Properties of Polybutyl Methacrylate.

The damping properties of polybutyl methacrylate (PBMA)/aromatic petroleum resin (C9) composite were investigated in this work. In particular, a trace of styrene (St) was introduced to copolymerize with PBMA to improve the compatibility between C9 and matrix. The structure of the copolymer, P(BMA-co-St), was characterized by FTIR and 1HNMR. The P(BMA-co-St)/C9 composites were tested by differencial scanning calorimetry (DSC), scanning electron microscopy (SEM) and dynamical mechanical analysis (DMA). DSC curves of all P(BMA-co-1wt%St)/C9 composites expressed only one glass transition temperature (Tg). SEM images showed that C9 had good compatibility with the matrix after St was introduced. DMA curves indicated that the addition of C9 had a positive effect on the damping properties of PBMA. The loss tangent (tanδ) peak moved to a higher temperature with the increment content of C9, and the effective damping temperature range increased significantly. The influence of aromatic resin C9 and aliphatic resin (C5) on PBMA damping performance was compared. It was further shown that C9 with benzene ring effectively improved the damping performance of PBMA.

Theoretical studies on the purine radical induced purine-purine type intrastrand cross-links.

At the density functional theory (DFT) level, addition reactions between the guanine-8-yl radical and its 3'/5' neighboring purine deoxynucleosides forming the purine-purine type intrastrand cross-links were studied. It is found that addition of the guanine-8-yl radical to the C8 site of its 5' neighboring deoxyguanosine or deoxyadenosine is a two-step reaction consisting of a structurally relatively unfavourable conformational transformation step, while the corresponding 3' C8 addition is straightforward and kinetically more efficient. The 3' C8 preference of the guanine-8-yl radical additions indicates the existence of an obvious sequence effect, which is completely opposite to that observed in the formation of pyrimidine radicals induced DNA intrastrand cross-links. The detrimental effects from steric hindrance and stabilizing weak interactions make these addition reactions markedly suppressed in double stranded DNA. This work broadens our knowledge about the possible types of DNA intrastrand cross-links.

Crystal Structure of C5b-6 Suggests Structural Basis for Priming Assembly of the Membrane Attack Complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.

Lifetimes and Reaction Pathways of Guanine Radical Cations and Neutral Guanine Radicals in an Oligonucleotide in Aqueous Solutions

The exposure of guanine in the oligonucleotide 5'-d(TCGCT) to one-electron oxidants leads initially to the formation of the guanine radical cation G(•+), its deptotonation product G(-H)(•), and, ultimately, various two- and four-electron oxidation products via pathways that depend on the oxidants and reaction conditions. We utilized single or successive multiple laser pulses (308 nm, 1 Hz rate) to generate the oxidants CO(3)(•-) and SO(4)(•-) (via the photolysis of S(2)O(8)(2-) in aqueous solutions in the presence and absence of bicarbonate, respectively) at concentrations/pulse that were ∼20-fold lower than the concentration of 5'-d(TCGCT). Time-resolved absorption spectroscopy measurements following single-pulse excitation show that the G(•+) radical (pK(a) = 3.9) can be observed only at low pH and is hydrated within 3 ms at pH 2.5, thus forming the two-electron oxidation product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single pulse excitation, the principal reactive intermediate is G(-H)(•), which, at best, reacts only slowly with H(2)O and lives for ∼70 ms in the absence of oxidants/other radicals to form base sequence-dependent intrastrand cross-links via the nucleophilic addition of N3-thymidine to C8-guanine (5'-G*CT* and 5'-T*CG*). Alternatively, G(-H)(•) can be oxidized further by reaction with CO(3)(•-), generating the two-electron oxidation products 8-oxoG (C8 addition) and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), appear only after a second (or more) laser pulse. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses.

Application of mixture topological index method to predict the dynamic viscosity of the hypothetical acidic oils-based biodiesel fuels

This work proposed a method to predict the dynamic viscosity values of the hypothetical acidic oils-based biodiesel fuels. A topological index that can formulate the molecular structural information of fatty acid methyl ester (FAME) components into dimensionless form was built from the integration and modification of the distance matrix and adjacency matrix of the molecular structure. Results show that both the caprylic acid (C8:0) and linoleic acid (C18:2) are beneficial components for reducing the biodiesel fuel’s dynamic viscosity. Nine hypothetical acidic oils-based biodiesel fuels were made from the mixing of C8:0 or C18:2 with one vegetable oil at the same mass ratio, including peanut, canola, coconut, palm, soybean, corn, rapeseed, sunflower and cottonseed oils, and the dynamic viscosity values of them were predicted. The dynamic viscosity values were ranged at 0.90–2.13 mPa s (with the addition of C8:0) and 2.83–4.07 mPa s (with the addition of C18:2). This investigation is expected to provide some suggestions for the selection of some components that are beneficial for reducing the product’s dynamic viscosity, so that the atomization characteristic of a low-quality feed can be improved by adding some suitable components into it.

Biomechanical analysis of Goel technique for C1–2 fusion

The Goel technique, in which C1-2 intraarticular spacers are used, may be performed to restore stability to a disrupted atlantoaxial complex in conjunction with the Harms technique of placing polyaxial screws and bilateral rods. However, it has yet to be determined biomechanically whether the addition of the C1-2 joint spacers increases the multiaxial rigidity of the fixation construct. The goal of this study was to quantify changes in multiaxial rigidity of the combined Goel-Harms technique with the addition of C1-2 intraarticular spacers. Seven cadaveric cervical spines (occiput-C2) were submitted to nondestructive flexion-extension, lateral bending, and axial rotation tests in a material testing machine spine tester. The authors applied 1.5 Nm at a rate of 0.1 Nm/second and held it constant for 10 seconds. The specimens were loaded 3 times, and data were collected on the third cycle. Testing of the specimens was performed for the following groups: 1) intact (I); 2) with the addition of C-1 lateral mass/C-2 pedicle screws and rod system (I+SR); 3) with C1-2 joint capsule incision, decortication (2 mm on top and bottom of each joint [that is, the C-1 and C-2 surface) and addition of bilateral C1-2 intraarticular spacers at C1-2 junction to the screws and rods (I+SR+C); 4) after removal of the posterior rods and only the bilateral spacers in place (I+C); 5) after removal of spacers and further destabilization with simulated odontoidectomy for a completely destabilized case (D); 6) with addition of posterior rods to the destabilized case (D+SR); and 7) with addition of bilateral C1-2 intraarticular spacers at C1-2 junction to the destabilized case (D+SR+C). The motion of C-1 was measured by a 3D motion tracking system and the motion of C-2 was measured by the rotational sensor of the testing system. The range of motion (ROM) and neutral zone (NZ) across C-1 and C-2 were evaluated. For the intact spine test groups, the addition of screws/rods (I+SR) and screws/rods/cages (I+SR+C) significantly reduced ROM and NZ compared with the intact spine (I) for flexion-extension and axial rotation (p < 0.05) but not lateral bending (p > 0.05). The 2 groups were not significantly different from each other in any bending mode for ROM and NZ, but in the destabilized condition the addition of screws/rods (D+SR) and screws/rods/cages (D+SR+C) significantly reduced ROM and NZ compared with the destabilized spine (D) in all bending modes (p < 0.05). Furthermore, the addition of the C1-2 intraarticular spacers (D+SR+C) significantly reduced ROM (flexion-extension and axial rotation) and NZ (lateral bending) compared with the screws and rods alone (D+SR). Study result indicated that both the Goel and Harms techniques alone and with the addition of the C1-2 intraarticular spacers to the Goel-Harms technique are advantageous for stabilizing the atlantoaxial segment. The Goel technique combined with placement of a screw/rod construct appears to result in additional construct rigidity beyond the screw/rod technique and appears to be more useful in very unstable cases.

Complement Inhibitor of C5 Activation from the Soft Tick Ornithodoros moubata

Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.

Effects of medium‐chain fatty acid‐cyclodextrin complexes on ruminal methane production in vitro

ABSTRACTThe purpose of the present study was to evaluate effects of medium‐chain fatty acid‐cyclodextrin (CD) complexes on ruminal methane and volatile fatty acid production, and protozoal activity in vitro. Medium‐chain fatty acid‐CDs used in this study were caprylic acid (C8)‐αCD or ‐βCD, capric acid (C10)‐αCD or ‐βCD, and lauric acid (C12)‐αCD or ‐βCD. A 60‐mL of diluted rumen fluid was incubated anaerobically at 38°C for 6 h with the addition of the complex (10–40 mg as fatty acid). Each of the fatty acid‐CDs reduced the number of protozoa, with the order C10 &gt; C12 &gt; C8, and βCD complexes were more effective than αCD complexes. Molar proportions of acetic acid remained unchanged with the addition of fatty acid‐CD, while that of propionic acid increased, being significant for C8‐αCD and βCD, and C10‐αCD and βCD (P &lt; 0.05). Hydrogen production decreased by about 70% of control with the addition of 40 mg of C8 and C10‐CD, on the other hand, it tended to increase with the addition of C12‐CD in both αCD and βCD. Methane production decreased by about 20% with the addition of 40 mg of complexes, except for C10‐βCD, which significantly reduced methane production by about 60%. In conclusion, the addition of C8 or C10‐CD to ruminant diets may be effective in reducing methane production.

Distinct effects of ceramide-generating pathways in prostate adenocarcinoma cells.

Ceramide, generated by the hydrolysis of sphingomyelin, mediates the actions of several cytokines such as tumour necrosis factor-alpha (TNF-alpha) interferon-gamma and interleukin-1beta (IL-1beta), including their inhibitory effect on tumour proliferation. We have evaluated the role of ceramide in the proliferation of prostate cancer by using the human prostate adenocarcinoma LNCaP cell line. Treatment of LNCaP cells with neutral or acidic sphingomyelinase or addition of C8- or C2-ceramide, two cell permeable analogues of endogenous ceramide, induced a profound inhibition of cell proliferation. This effect appeared after 24 h, was still present after 72 h of exposure to the drugs and exhibited concentration-dependency (10-200 and 5-200 mU ml(-1) for neutral and acidic sphingomyelinase, respectively, and 1-25 microM for C8-ceramide). The inhibitory effect on cell growth caused by neutral sphingomyelinase and ceramides was rapidly reversible as LNCaP cells rapidly regained their previous proliferation rate following withdrawal of the treatment. IL-1beta produced profound inhibition of LNCaP cell proliferation and caused enhanced ceramide formation. No clear features of apoptotic cell death were detectable by either oligonucleosome formation, cytofluorimetric analysis or nuclear staining following exposure of LNCaP cells to neutral sphingomyelinase, ceramide or IL-1beta. However, clear changes in LNCaP cell cycle distribution were detectable following these treatments. In contrast, treatment with acidic sphingomyelinase or TNF-alpha induced apoptotic death detectable by flow cytometric analysis and bisbenzimide staining. In conclusion, our data demonstrate that preferential activation of distinct enzymatic pathways by cytokines may lead to different outcomes in the viability of LNCaP cells.

Crystallization of vacuum-evaporated Se studied by near infrared microscope

The crystallization of vacuum-evaporated Se can be greatly affected by impurities present either in the Se source or on the substrate surface [1-3]. The presence of oxygen (in the form of SeO2) in the Se source was found to enhance Se crystallization [1], due to formation of new nucleation sites on the substrate surface. In contrast, the addition of C1, As, and Te alloys in the Se source was observed to inhibit Se crystallization, due to deactivation of chain growth on the crystal surface [2, 3]. Crystallites were also observed in amorphous Se films after ageing in air, even at room temperature and without illumination (the activation energy for the crystallization of Se is about 1 eV) [4-7]. Along with reorganization of the Se molecules [6, 7], the adsorption of oxygen and water impurities on the Se surface is believed to play an important role. We have reported [8] that the crystallization of vacuum-evaporated high purity Se on glass, ITO (indium tin oxide), and PC (polycarbonate) substrates can be greatly enhanced and the structure of the crystallites modified if the substrates have been soaked in water prior to Se deposition. Crystallization was further increased by heating the Se films in air, the rate of crystal growth being faster than that in the vacuum chamber. These observations lead to the conclusion that water impurities, initially absorbed in the substrate during soaking, can 'enhance crystallization of Se by forming additional nucleation sites on the substrate surface. To further investigate this effect, and in particular, to effectively eliminate water molecules from the substrates, we examine the influence of several parameters affecting water content in the substrates on the crystallization of Se. These parameters include water soaking temperature and heating and pumping substrates in the vacuum chamber. Glas, ITO and PC substrates [8] were cleaned using reagent grade isopropanol and dried with a jet of dry pre-purified nitrogen. Some of the substrates were soaked in de-ionized water for 1 min and again

Ultrastructural studies of complement mediated cell death: a biological reaction model to plasma membrane injury.

Complement-mediated nucleated cell death has been shown to be independent of colloid-osmotic swelling. In contrast, other factors (e.g. Ca2+ influx) are of importance in the induction of cell death. In this communication, the sequential morphological features of complement-mediated cell injury have been studied by electron microscopy and compared with biochemical data (ATP content and LDH release). It was observed that immediately after C5b-8 lesion formation, although the overall cell, morphology is well preserved, the mitochondria display an "ultracondensed" appearance. Upon addition of C9, the mitochondria remain initially condensed, but swell progressively with final formation of flocculent densities. The nuclei become progressively edematous, with concurrent disappearance of heterochromatin. The nucleoli lose their associated chromatin and display segregation of their components with formation of markedly electron-dense filamentous deposits. The nuclear envelope remains initially intact, but subsequently progressive dilatation of the associated perinuclear RER cisterna and distention of the nuclear pores associated with leakage of chromatin into the cytoplasm are seen. The larger cell organelles (including mitochondria, ER, Golgi apparatus, etc.) become clustered around the nucleus, concurrently with marked edema of the outer cytoplasm and bleb formation. The RER cisternae become dilated, whereas the Golgi complex disappears. Relatively early on the plasma membrane shows breaks in continuity. The pattern of these changes--potentially related to Ca2+ influx, ATP efflux and overall metabolic depletion--corresponds to the previously described model of cell reaction to injury, confirming the dynamic nature of the process. The morphology of cell death in this model shares some features, e.g., the nucleolar changes, with "apoptosis" (programmed cell death). However, the overall pattern appears to correspond more to "necrosis," characterized by loss of volume control and mitochondrial abnormalities.

Mechanism of killing of Giardia lamblia trophozoites by complement.

Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.

The membrane attack complex in complement-mediated glomerular epithelial cell injury: formation and stability of C5b-9 and C5b-7 in rat membranous nephropathy.

Using a model of rat membranous nephropathy (MN), we examined the relationship between the development of glomerular epithelial cell injury and the formation and stability of the membrane attack complex (MAC) of complement. Isolated rat kidneys were perfused with buffered bovine albumin (BSA) or various plasmas (complement source). Kidneys containing nephritogenic amounts of complement-fixing sheep antibody to glomerular epithelial antigens (aFx1A) perfused with BSA (n = 5), and normal kidneys perfused with normal human plasma in BSA (50% v/v, n = 6) excreted 0.30 +/- 0.02 mg protein/min/g during 90 min perfusion (control groups). When normal plasma was added to the perfusate of aFx1A kidneys at concentrations of 12.5, 25, and 50% v/v, protein excretion rose in a time- and concentration-dependent manner. Perfusions with 25% plasma resulted in baseline proteinuria from 0 to 20 min that increased to 2.8 +/- 0.9 mg/min/g at 20 to 40 min and 8.6 +/- 2.1 at 40 to 60 min (n = 4, p less than 0.01 vs control groups). Removal of plasma at 20 min did not prevent this rise in protein excretion (3.9 +/- 2.4 and 5.8 +/- 2.6 mg/min/g at 30 to 40 and 55 to 65 min respectively, p less than 0.01, n = 4). Perfusion of aFx1A kidneys with C8-deficient (C8D) human plasma (25% v/v, n = 4) or C6D rabbit serum (25% v/v, n = 2) independently produced low levels of proteinuria comparable with BSA, but in combination, the two reagents restored enhanced protein excretion (n = 2). In aFx1A kidneys containing C5b-7, addition of C8 and C9 (C6D serum) after intervals of 20, 60, or 90 min immediately reconstituted heavy proteinuria. Thus, the magnitude of MAC-induced glomerular epithelial injury in rat MN is related to the complement dose. Altered glomerular permeability is delayed with respect to the onset of complement activation. Once sufficient C5b-9 is formed, proteinuria can develop despite cessation of new MAC assembly, implying that C5b-9 persists after formation. Moreover, the C5b-7 MAC intermediate is not eliminated rapidly in this model.

Fluid-phase assembly of the membrane attack complex of complement.

The dynamics and protein stoichiometry of the fluid-phase assembly of the membrane attack complex of complement were characterized by using light-scattering intensity measurements. The assembly proceeded in an ordered manner with generation of stable and highly reproducible intermediates. In the absence of phospholipid or C8, mixtures of C5b-6 and C7 self-associated to fluid phase-C5b-7 which had a weight-average molecular weight of (4.1 +/- 0.2) X 10(6). This corresponded to an average of nine C5b-7 complexes per particle. The particles appeared heterodisperse on sucrose gradients with S20,W values ranging from 21 to 39 S. Addition of C8 and C9 caused no further aggregation or disassembly of the particles. When excess C8 was added to the aggregated C5b-7, the ratio of C8 incorporated per C5b-7 moiety was 0.98 +/- 0.03. At saturating levels of C9, the C9/C5b-8 ratio in the particles was 7.2 +/- 0.6. Incorporation of C8 caused a small increase in the Z-averaged particle diffusion coefficient [(9.9-10.3) X 10(-8) cm2/s], indicating that it added in a manner that "filled in the gaps" in the C5b-7 particles. C9 caused only small decreases in the particle diffusion coefficient and substantially decreased the f/fmin ratio. The time course for C9 incorporation into fluid phase-C5b-8 indicated an initial rapid phase followed by a slow phase. The rapid phase corresponded to the incorporation of about one C9 for every two C5b-8 complexes. This suggested that one C9 binding site was accessible on about half of the C5b-8 complexes. This may imply that only about half of the C5b-8 complexes were capable of C9 polymerization so that the ratio of C9 incorporated per functional C5b-8 was (14 +/- 2)/1. The initial velocity of the slow phase of C9 addition gave an activation energy of 37 kcal/mol. The activation energy for C5b-8-independent polymerization of C9 had a similar value of 41 kcal/mol. Light-scattering intensity measurements seemed to be a highly reliable method for quantitative characterization of the fluid-phase assembly.

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9.

The fluorescent potentiometric indicator diS-C3-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts--bearing membrane-assembled C5b67 complexes--were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (-7 mV) and ghosts (O mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential--and the consequent onset of hemolysis--is accelerated by raising external [K+], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K+] or [Na+], no change in membrane potential (from O mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from O mV by selectively increasing the K+ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was less than 5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmembrane equilibration of Na+ and K+ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (Em) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K+ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the near-equilibrium condition of potassium-containing red cells when suspended at high external K+. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.

Antibody-independent and -dependent opsonization of group B Streptococcus requires the first component of complement C1.

The role of the classical complement pathway and specifically the first component, C1 in antibody-independent opsonization of type Ia group B Streptococcus (GBS) was investigated. For these studies a radiolabeled bacterial uptake assay was developed that was dependent on time and bacterial concentration and that required an intact classical complement pathway. To directly investigate the role of C1 in opsonization of type Ia GBS, C1 was isolated by chromatography on an immunoglobulin G (IgG) affinity column and further purified by molecular sieve chromatography on an Ultrogel AcA 22 column. When normal human serum was absorbed with 10(9) CFU of type Ia or III GBS, the serum opsonic capacity diminished (33 to 34%) for type Ia GBS compared with unadsorbed serum. Preincubation of the bacteria with purified C1 (10(4)U of C1 per ml) restored the opsonizing capacity of the adsorbed serum. A C1-depleted serum was prepared from the nonadherent fractions of the CH-sepharose 4B IgG column which only contained 5 U of C1 per ml. Substitution of C1-depleted reagent for normal serum in the uptake assay resulted in dramatic decreases in the opsonization of type Ia GBS, but opsonization could be restored by preincubation of the bacteria with purified C1. Heat-inactivated C1 depleted serum did not support opsonization of type Ia GBS, even with the addition of C1. Preincubation of type Ia GBS with heat-inactivated hyperimmune sera did not result in opsonization of type Ia GBS in the presence of C1-depleted serum. However, opsonization could be restored by the addition of C1, and the effects of C1 and antibody were additive. These results indicate the critical role of C1 in direct activation of the classical complement pathway by type Ia GBS and in antibody-mediated opsonization of the bacteria.

Effect of fibronectin on the haemolytic activity of complement

Sheep erythrocytes were coupled with trinitophenyl sulphonate, sensitized with anti-TNP (or-DNP) IgM monoclonal antibodies, and exposed to components of the classical pathway of complement activation. When human fibronectin (FN) was added after C1q, but before addition of C1r and C1s (subunits of the first complement component), inhibition of haemolytic activity was observed which was strictly dependent upon the dose of FN. When FN was added after addition of C1 (reconstituted from C1q, C1r and C1s), the haemolytic activity of complement was not affected by the presence of FN. These data suggest that FN binds on C1q by interfering with C1r and C1s fixation. In addition, FN was unable to displace the activated subcomponents (C1r and C1s) from their binding site on C1q. When using other systems (sheep erythrocytes sensitized with anti-Forssman IgM monoclonal antibodies), the quantity of FN required to inhibit complement haemolytic activity was greater than in the TNP system. In normal plasma, there is a 50-fold excess of FN compared to free C1q.

The attack phase of human complement: differentiation between membrane binding and complex formation by the detection of neoantigen expression in situ. A morphometric immunoferritin study.

C5-9 neoantigen was found by morphometric immunoferritin staining on the membrane of guinea pig erythrocytes lyzed by reactive lysis C5b6 or acid-activated C5 + C6 and C7, C8, and C9. Neoantigen first appeared at the C7 step, increasing with C8, and decreasing after the addition of C9. The incubation of E with C5-C8 or C5-C9 at 0 degrees C resulted in EC5-8 without neoantigen. Reincubation of these cells after washing at 37 degrees C for 5 min led to neoantigen expression. An energy-requiring step is postulated after binding of C5-C8 and needed for C9 binding and lysis.

Soluble C5b-9 complex of guinea pig complement: Demonstration of its heterogeneity and the mechanism of its C9 hemolytic activity as transfer of reversibly bound C9 molecules from the complex

Doubly radiolabeled soluble complex of the late acting components (SC5b-9) was formed by activating guinea pig complement (C)† serum mixed with C5 and C9 each labeled with either 125I or 131I with zymosan. It was isolated by sequential gel filtrations and sucrose density gradient centrifugation. Fractions of the complex obtained by centrifugation were heterogeneous in size, composition and hemolytic activity as C9. The heavier fractions contained four to six molecules of C9 per C5b molecule. The lighter fractions contained two to three C9 molecules to one C5b molecule and had stronger C9 hemolytic activity. On recentrifugation on the same sucrose density gradient, the factions moved to the same positions as on the first centrifugation. Guinea pig SC5b-9 complex bound to the surface of EA, EAC-3, EAC-5, EAC-6, EAC-7 and EAC-8 in similar amounts. Therefore, combination of the complex with these intermediate cells seems to be nonspecific. EAC-7 cells bound to the complex could be lysed by addition of C8. Lighter, hemolytically more active complex purified by recentrifugation was stable when incubated alone in buffer. But when it was incubated with free C9, it released radiolabeled C9, the amount released increasing with increase in the amount of free C9 added to a maximum of 10 to 15% of the C9 in the complex. A minute part of the complex was converted to heavier complex by incorporation of additional C9, but most of the complex did not change in size. Therefore, it did not seem to be an uncompleted complex to be saturated with more C9. The C9 molecules released from the complex were in an active form and could be reincorporatd into SC5b-9 when they were treated with zymosan in fresh guinea pig serum. It is concluded that the C9 hemolytic activity of guinea pig SC5b-9 is exerted by transfer of reversibly bound C9 molecules in the complex to C5b-8 sites on EAC-8 cells, not by the action of the complex in toto.