Humoral immune response against hepatitis C virus

Antibodies are in several instances a reliable marker indicating vigorous immune response against infectious agents and in several viral diseases presence in the blood of specific anti-viral antibodies indicates an effective protection. However, this is not always true. For example, in the case of hepatitis C virus (HCV) an important human pathogen considered the causative agent of the nonA- nonB hepatitis, in spite of an intense antibody response there is no protection against a new infection and in the majority of infected individuals the virus overcomes host defences establishing a persistent infection. Here we describe how the dissection of the humoral immune response against HCV glycoprotein E2 of infected patients was useful for a better comprehension of the virus-host interplay. Cross-reactive antibodies directed against E2 are produced by the HCV-infected patient, but not all of them are protective, and some could even result to be detrimental for the patient. The cross-reactive anti-HCV/E2 humoral antibody response is complex and not necessarily completely beneficial to the host.     

Paralytic peptide activates insect humoral immune response via epidermal growth factor receptor

Paralytic peptide (PP) activates innate immunity of silkworm Bombyx mori, inducing production of anti-microbial peptides (AMPs) and phagocytosis-related proteins; however the signal pathways of PP-dependent immune responses are not clear. In present study, we characterized BmE cells as a PP-responsive cell line by examining the expression of AMP genes and activation of p38 mitogen-activated protein kinase (p38 MAPK) under PP stimulation, and we also found PP directly binds to BmE cell membrane. Then we found that PP-dependent expression of AMP genes is suppressed by tyrosine kinase inhibitor (genistein) both in BmE cells and in fat body of silkworm larvae. Moreover, the specific tyrosine kinase epidermal growth factor receptor (EGFR) inhibitor (AG1478) attenuates PP-induced expression of AMP genes in BmE cells and fat body of silkworm and RNA interference (RNAi) to BmEGFR also suppresses PP-induced expression of AMP genes. Furthermore, the PP-induced p38 MAPK phosphorylation is inhibited by AG1478. Our results suggest that BmE cells can be used as a cell model to investigate the signal pathway of PP-dependent humoral immune response and receptor tyrosine kinase EGFR/p38 MAPK pathway is involved in the production of AMPs induced by PP.     

Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii, which is widely distributed in nature, and presents a saprophytic mycelial form on plant debris and soil. The immunological mechanisms involved in the prevention and control of sporotrichosis are not yet fully understood. In this study, mice were studied after infection with Sporothrix schenckii. In the first week after infection, fungal loading increased and thence decreased drastically 14 days after infection. Analysis by immunoblotting showed that the sera of all mice tested had antibodies reacting only with a 70 kDa antigen, with predominance of IgG1 and IgG3. Taken together, our results show that antigens from S. schenckii induced a specific humoral response in infected mice.     

Humoral immune response in mice over-expressing or deficient in growth hormone

Effects of growth hormone (GH) levels on the humoral immune response were investigated in metallothionein I (MT)-bovine (b) GH-transgenic (tg) and GH-deficient Ames dwarf (Prop1 df(-/-)) mice. Four-month-old mice were given primary and secondary injections of either normal saline or tetanus toxoid (TT) to induce specific antibody (Ab) production. MT-bGH-tg mice with high peripheral levels of bGH produced less TT-specific Ab than normal nontransgenic (Ntg) littermates, df, or nondwarf (Ndf) control mice. Titers reached maximum levels at 3-4 weeks post-primary immunization (PPI) and declined gradually through 24 weeks PPI in all groups of mice. Peripheral CD4(+) and CD8(+) T cell populations were significantly lower in tg than in Ntg, df, or Ndf mice. No significant differences were found in B cell numbers between tg, Ntg, or df mice. T helper 2 (Th2) cell populations were significantly greater in df mice compared to Ntg control mice. No significant differences were found in CD4(+):CD8(+) T cell ratios, interleukin (IL)-4 concentrations or interferon (IFN)-gamma levels between tg,Ntg, df, and Ndf mice. No patterns of significant sexual dimorphism were found for any of the immune parameters studied. Elevated levels of corticosterone were investigated as a possible immunosuppressant mechanism responsible for low Ab responses in the tg mice. Ab production was not enhanced by decreasing corticosterone in tg mice. Thus, high endogenous GH levels inhibit specific Ab production and peripheral T cell populations but not peripheral B cell numbers, Th2 cell populations, or IL-4 or IFN-gamma production. Elevated corticosterone levels do not appear to be responsible for suppressed humoral immune responses. Low levels of endogenous GH do not inhibit specific Ab production but may contribute to increased peripheral Th2 cell numbers.     

Stimulation by epidermal growth factor of phospholipid methyltransferase in isolated rat hepatocytes

Epidermal growth factor produces a time- and dose-dependent activation of phospholipid methyltransferase activity in hepatocytes isolated from juvenile and mature hepatectomized rats. This treatment however has no effect with hepatocytes isolated from mature or laparotomized rats. Dansylcadaverine (50μM), an inhibitor of receptor-mediated internalization of epidermal growth factor, has no effect on basal phospholipid methyltransferase but inhibits the stimulation of this enzyme by epidermal growth factor.

These results indicate a possible role of phospholipid methylation during liver proliferation.     

大花旋蒴苣苔对脱水与复水的生理响应

本试验以陕西、四川(下称西部)、安徽和浙江(下称东部)4省区大花旋蒴苣苔(Boea clarkeana Hemsl.)的离体叶片为材料,通过对不同脱水时间(0、4、8、12、24和48 h)和脱水48 h后不同复水时间(4、8、12、24和48 h)条件下大花旋蒴苣苔生理指标变化的研究,比较了中国东部和西部地区大花旋蒴苣苔的耐脱水特性。结果表明:4种群大花旋蒴苣苔的相对含水量在脱水过程中呈显著下降趋势(P0.05);相对含水量下降的总体趋势表现为浙江种群安徽种群四川种群陕西种群;在复水过程中,4种群相对含水量基本恢复到实验前水平;超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)3种抗氧化酶活性以及MDA、脯氨酸、可溶性糖和可溶性蛋白的含量在脱水过程中均表呈显著上升趋势(P0.05),在复水过程中均呈显著下降趋势(P0.05)。通过对4种群生理指标的比较研究发现,4种群的耐脱水性整体表现为陕西种群四川种群安徽种群浙江种群,即西部种群的耐旱性强于东部地区。     

Production of RNA by a polymerase protein encapsulated within phospholipid vesicles

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.Abbreviations ADP adenosine diphosphate - DMPC dimyristoyl phosphatidylcholine - EDTA ethylenediaminetetraacetic acid - LUV large unilamellar vesicle - MLV multilamellar vesicle - PAGE polyacrylamide gel electrophoresis - PNPase or PNP polynucleotide phosphorylase - SUV small unilamellar vesicle Correspondence to.: A.C. Chakrabarti     

Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.     

Fluid balance in exercise dehydration and rehydration with different glucose-electrolyte drinks

After exercise dehydration (3% of body weight) the restoration of water and electrolyte balance was followed in 6 male subjects. During a 2 h rest period after exercise, a drink of one of four solutions was given as 9 X 300 ml portions at 15 min intervals: control (C-drink), high potassium (K-drink), high sodium (Na-drink) or high sugar (S-drink). An exercise test (submaximal and supramaximal work) was performed before dehydration and after rehydration. Dehydration reduced plasma volume by 16%, a process reversed on resting even before fluid ingestion began, due to release of water accumulated in the muscles during exercise. After 2 h rehydration, plasma volume was above the initial resting value with all 4 drinks. The final plasma volumes after the Na-drink (+14%) and C-drink (+9%) were significantly higher than after the K- and S-drinks. The Na-drink favoured filling of the extracellular compartment, whereas the K- and S-drinks favoured intracellular rehydration. In spite of the higher than normal plasma volume after rehydration, mean heart rate during the submaximal test was 10 bpm higher after rest and rehydration than in the initial test, and was not different between the drinks. The amount of work which could be performed in the supramaximal test (105% VO2max) was 20% less after exercise dehydration and subsequent rest and rehydration than before. This reduction was similar for all drinks, and may be due to a decreased muscle glycogen content (70% of initial) at the time of the second test.     

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.     

Effect of sterol structure on the partition of sterol between phospholipid vesicles of different composition

The partition of cholesterol analogues between dipalmitoylphosphatidylcholine and egg phosphatidylcholine vesicles was examined. Cholesterol, trans- and cis-22-dehydrocholesterols, and 24 alpha-ethyl,trans-22-dehydrocholesterol (stigmasterol) showed a preference from gel phase dipalmitoylphosphatidylcholine over fluid phase egg phosphatidylcholine at 37 degrees C. Within this group, the sterol concentration in DPPC relative to that in egg PC ranged from about 1.5 to 2.0. Cholesterol analogues with a 24 alpha-methyl or ethyl substituent (campesterol and beta-sitosterol, respectively) and cholestanol (dihydrocholesterol) distributed about equally between the two types of phospholipid. Thus, in this study involving two kinds of phospholipid and a small number of cholesterol analogues, there was no simple correlation between the sterol structure and its partition behavior. The combined results from studies on sterol partition behavior and on sterol interaction with individual phospholipids (Rujanavech, C., Henderson, P.A., and Silbert, D.F. (1986) J. Biol. Chem. 261, 7204-7214) provide an adequate basis to explain the different patterns of membrane lipid adaptation which accompany growth of LM cells on various cholesterol analogues (Rujanavech, C., and Silbert, D.F. (1986) J. Biol. Chem. 261, 7196-7203).     

Sequential appearance of epidermal growth factor in plasma membrane-associated and intracellular vesicles during endocytosis

Receptor-mediated internalization of epidermal growth factor (EGF) occurs by a process involving initially clathrin-coated pits on the cell surface and the subsequent formation of ligand-containing endosomes. Using a modified acid wash technique, cell surface-bound EGF was removed. Utilizing sucrose density centrifugation, the residual cell-associated EGF was separated into plasma membrane-associated and intracellular vesicle-associated forms. Using these procedures we have identified a transient form of cell-associated EGF that is still attached to the plasma membrane but not accessible to the extracellular fluid. This form of EGF appears to be the precursor for endosomic EGF. We suggest that this intermediate form represents the receptor-ligand complex shown by electronmicroscopy to be located in narrow-necked plasma membrane invaginations (Willingham, M. C., and Pastan, I. (1980) Cell 21, 67-77).     

The binding of factor Va to phospholipid vesicles

The analysis of free sulfhydryl groups in factor Va using dithiobis-(nitrobenzoic acid) (DTNB) indicated the presence of one accessible thiol in each of the two subunits of the cofactor. Intact factor Va contained one readily accessible sulfhydryl group under native conditions and approximately two such groups after denaturation. A comparison of the rate of modification of the accessible thiol in factor Va under native conditions to those observed with the isolated subunits indicated that the thiol present in component D of the cofactor was readily accessible to reaction with DTNB. Factor Va was reacted with the sulfhydryl-directed fluorophore N-(1-pyrene)maleimide, resulting in the concomitant loss of the accessible thiol with no detectable alteration in the activity of the cofactor. This fluorescent derivative of factor Va (Pyr-Va) was used to examine the binding of factor Va to phospholipid vesicles by fluorescence polarization. Fluorescence polarization of the pyrene moiety increased saturably when Pyr-Va was titrated with increasing concentrations of vesicles composed of phosphatidylcholine and phosphatidylserine (PS). Systematic analysis of the binding of Pyr-Va to PCPS (75% phosphatidylcholine, 25% PS) indicated that the binding interaction was characterized by a dissociation constant of 2.7 x 10(-9) M with 42 mol of PCPS bound per mol of Va at saturation. The data obtained by varying the PS content of the vesicles are consistent with the interpretation that the Va-combining site on the vesicle surface is composed of a discrete number of PS molecules. The binding of Pyr-Va to PCPS was independent of added calcium ion and could be reversed by the addition of unlabeled Va or isolated component E but not by component D. Analysis of the displacement curves indicated that native factor Va or isolated component E and Pyr-Va mutually excluded each other on the vesicle surface with identical affinities. Competition experiments conducted using component E digested by factor Xa or the isolated derivative peptides indicated that the cleavage of component E by factor Xa had no effect on the PCPS binding properties of this subunit. Further, the data obtained with the isolated peptides suggest that the lipid-binding domain of component E is present in the amino-terminal region of this subunit.     

Changes in phospholipid extractability and composition accompany mineralization of chicken growth plate cartilage matrix vesicles.

Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen.     

Binding of human factor VIII to phospholipid vesicles

Factor VIII, a protein cofactor involved in blood coagulation, functions in vitro on a phospholipid membrane surface to greatly increase the rate of factor X activation by factor IXa. Using gel filtration, rapid sedimentation, and resonance energy transfer we have studied the interaction of recombinant-derived human factor VIII with small and large unilamellar phospholipid vesicles composed of phosphatidylserine and phosphatidylcholine. Resonance energy transfer, from intrinsic fluorophores in factor VIII to dansyl-phosphatidylethanolamine incorporated into vesicles, has been adapted for quantitative equilibrium measurements. Factor VIII binds rapidly and reversibly to small and large vesicles. At 8 degrees C the interaction of factor VIII with small vesicles fits a simple bimolecular model with a KD of 2 nM and a phospholipid binding site defined by 180 phospholipid monomers. At 25 degrees C the binding of factor VIII to small vesicles containing 20% phosphatidylserine can be described by an apparent KD of 4 nM; the phospholipid/protein ratio at saturation was 170. Binding to large vesicles was demonstrated with a KD of 2 nM and a phospholipid/protein ratio at saturation of 385. Binding was dependent upon the phosphatidylserine mole fraction and was nonlinear from 0 to 30% phosphatidylserine content. A direct comparison of factor VIII and factor V binding indicated that the affinity of factor V to phospholipid vesicles was equivalent to that of factor VIII and that the phosphatidylserine requirement was lower. A model is proposed to explain the nonlinear phosphatidylserine dependence of binding for factor VIII.     

Humoral immune response to filarial antigens in chyluria

Humoral immune parameters like total immunoglobulins and specific antibody levels in serum were studied in filarial chyluria patients. Mean serum IgG was significantly reduced in this group compared to normal controls, while IgA and IgM levels remained comparable to controls. Anti-filarial antibody titre as measured by enzyme-linked immunosorbent assay also was significantly reduced. However, the total and specific IgE antibody titre was similar to that of controls. Specific IgE contents of the patients’ sera could be related to their microfilaraemic status.     

Ectodomain shedding-dependent transactivation of epidermal growth factor receptors in response to insulin-like growth factor type I

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R(-) cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R(+) cells), stimulation of R(+) cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R(-) cells. The R(-) cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R(+) cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner.     

两种不同生境苦苣苔科植物的复苏特性及其对水分的光合和生理响应

复苏植物可以耐受极度干旱的环境,脱水至10％相对水分含量后仍然可以复苏.苦苣苔科植物包含有较多复苏植物,不同类群的复苏机理可能存在差异.该文选择分布在亚热带和温带石灰岩地区的锈色蛛毛苣苔(Paraboea rufescens)和心叶马铃苣苔(Oreocharis cordatula)两种苦苣苔科植物,并对这两个物种的叶...