Effect of latent cytomegalovirus infection on the antibody response to influenza vaccination: a systematic review and meta-analysis.

Latent infection with cytomegalovirus (CMV) is thought to accelerate aging of the immune system. With age, influenza vaccine responses are impaired. Although several studies investigated the effect of CMV infection on antibody responses to influenza vaccination, this led to contradicting conclusions. Therefore, we investigated the relation between CMV infection and the antibody response to influenza vaccination by performing a systematic review and meta-analysis. All studies on the antibody response to influenza vaccination in association with CMV infection were included (n = 17). The following outcome variables were extracted: (a) the geometric mean titer pre-/post-vaccination ratio (GMR) per CMV serostatus group, and in addition (b) the percentage of subjects with a response per CMV serostatus group and (c) the association between influenza- and CMV-specific antibody titers. The influenza-specific GMR revealed no clear evidence for an effect of CMV seropositivity on the influenza vaccine response in young or old individuals. Meta-analysis of the response rate to influenza vaccination showed a non-significant trend towards a negative effect of CMV seropositivity. However, funnel plot analysis suggests that this is a consequence of publication bias. A weak negative association between CMV antibody titers and influenza antibody titers was reported in several studies, but associations could not be analyzed systematically due to the variety of outcome variables. In conclusion, by systematically integrating the available studies, we show that there is no unequivocal evidence that latent CMV infection affects the influenza antibody response to vaccination. Further studies, including the level of CMV antibodies, are required to settle on the potential influence of latent CMV infection on the influenza vaccine response.

The use of monoclonal antibodies to study the structure and function of cytochrome f.

Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb-ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b6/f complex but MAb-ED4 did not. These results indicate that MAb-JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b6/f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were L-D-Q-P-L-T-S-N. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane-spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were N-I-L-V-I-G-P-V. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154.

Is functional manganese involved in hydrogen-peroxide-stimulated anomalous oxygen evolution in CACl2-washed photosystem II membranes?

When detergent-derived photosystem II (PSII) membranes are treated with CaCl2 to remove the three extrinsic proteins associated with the O2-evolving complex, the resulting membranes (CaPSII) can still catalyze water oxidation if sufficient Ca(2+) and Cl(-) are present. When CaPSII membranes are exposed to single turnover flashes on an O2 rate electrode, anomalous O2 is produced by the first two flashes. The addition of catalase to the membrane suspension completely inhibits O2 produced by the first two flashes, but not by subsequent flashes. Exogenous H2O2 stimulates anomalous O2 production by the first few flashes in CaPSII membranes, but not in control PSII membranes. Diuron (DCMU) does not inhibit H2O2-stimulated O2 production by the first flash. However, it does inhibit the O2 yield of all subsequent flashes, indicating that all flash-induced O2 signals in CaPSII membranes are dependent on photosystem II electron transport. H2O2 stimulation of O2 yields is inhibited in Tris-, heat-, and EDTA-(ethylenediaminetetraacetic acid)-treated CaPSII. In the presence of high salt, H2O2 (but not EDTA) treatment of CaPSII, extracts Mn functional in normal photosynthetic O2 evolution. The addition of exogenous Mn(2+) reconstitutes anomalous O2 production in Tris-and H2O2/EDTA-treated CaPSII preparations but only in the presence of H2O2. Anomalous H2O2-stimulated O2 production can be observed both with a Clark electrode (steady state) and an O2 rate electrode (flash sequence). The mechanism involves electron donation from H2O2, mediated by free Mn(2+), to PSII, and the 33-kDa extrinsic protein under some conditions can block this process. Since H2O2 can remove functional Mn from CaPSII membranes, its presence can convert functional Mn to the Mn(2+) mediator state required for anomalous O2 production. EDTA binds Mn in CaPSII disrupted by H2O2 and prevents anomalous O2 evolution.

Role of non-kinetochore microtubules in spindle elongation in mitotic PtK1 cells.

PtK1 metaphase cells were treated with varying concentrations of nocodazole to reduce spindle microtubule number and spindle length. The range of concentrations employed reduced spindle length from approximately 47% to 82% of the original pole-pole distance. Electron microscopy of cells treated with the lowest concentration of nocodazole employed (0.01 microgram/ml) showed a small decrease in the number of non-kinetochore microtubules (nkMTs), particularly evident in the astral region, with no significant effect on kinetochore microtubule number. Metaphase cells treated with 1 microgram/ml nocodazole for 2 min demonstrated a reduction in spindle length and loss of most non-kinetochore microtubules with little effect on the number and arrangement of the kinetochore class of microtubules. Following nocodazole treatment, the cells were perfused with 0.5 M sucrose dissolved in tissue culture medium, a treatment which has previously been shown to induce spindle elongation in metaphase cells. In cells where nocodazole effected a large decrease in non-kinetochore microtubule number with a concomitant decrease in spindle length, sucrose treatment had a reduced effect in inducing spindle elongation. In cells treated with lower concentrations of nocodazole, where numerous non-kinetochore microtubules remained, sucrose had a greater effect in inducing spindle elongation. These data suggest that the non-kinetochore population of microtubules is responsible for the extent of sucrose-induced spindle elongation. An explanation of these data is provided which suggests that the role of non-kinetochore microtubules is to trap energy in the developing spindle, such that it can be used to separate spindle poles during anaphase B.

Transverse heterogeneity in lipid fluidity in spinach thylakoids, photosystem II preparations, and thylakoid galactolipid vesicles.

We have used three doxyl stearic acid spin labels to study the transverse hetero-geneity in lipid fluidity in thylakoids, photosystem II (PS II) preparations, and thylakoid galactolipid vesicles. This comparative study shows that spin labels incorporated into the membrane of the PS II preparation experience far more immobilization than do the same spin labels incorporated into either thylakoids or vesicles prepared from the polar lipids extracted from thylakoids. The spin label immobilization found in the PS II preparation is manifest even near the center of the bilayer, where lipid mobility is normally at its maximum. Analysis of the lipid content of the PS II preparation, relative to chlorophyll, suggests that the PS II preparation may be lipid depleted. This lipid depletion could explain the results presented. However, electron microscopy [Dunahay et al. (1984) Biochim. Biophys. Acta 764:179-193] has not indicated that major delipidation has occurred, and so it remains possible that the immobilization found in the PS II preparation is due primarily to the normal (but close) juxtaposition of adjacent PS II complexes and the cooperative immobilization of their surrounding lipids. Based on the results presented, we conclude that highly mobile lipids are not required for oxygen evolution, the primary photochemistry or the secondary reduction of exogenously added quinones. Unfortunately, the relationship between the plastoquinone pool and the fluidity of the membrane in the PS II preparation remains ambiguous.

The use of polyclonal antibodies to identify peptides exposed on the stroma side of the spinach thylakoid.

We have raised polyclonal antibodies against an oxygen-evolving photosystem II preparation. Western Blot analysis of the whole serum revaals antibodies specific for at least 15 Coomassie visible bands ranging from 59 to 11 kDa. These antibodies are specific for proteins located on both sides of the membrane. Included are antibodies specific for Tris-removable peptides (33, 25 and 18 kda), which are thought to be exposed on the lumen surface of the PS II complex. Since the whole serum agglutinates thylakoids, antibodies specific for the stroma side of the PS II complex are also present. A sub-population of antibodies can be isolated by allowing the antibodies in whole serum to bind to EDTA-treated thylakoid membranes. The antibodies which specifically bind are cross-reactive with peptides with Mr of 59, 57, 34, 28, 27, 26, and 23 kDa. Our data indicate that these peptides have antigenic determinants exposed on the stroma side of the thylakoid membrane.

Sucrose-induced spindle elongation in mitotic PtK-1 cells.

Brief treatment of mitotic metaphase and anaphase PtK-1 cells with tissue culture medium containing 0.5 M sucrose resulted in spindle elongation without chromosome motion. Spindle birefringence also changed from a uniform appearance to one of highly birefringent bundles. Electron microscopic analysis indicated these birefringent bundles were composed of tightly packed arrays of spindle microtubules. No kinetochores could be seen following a 10 min sucrose treatment. Upon removal of sucrose, metaphase spindles returned to pretreatment lengths and the normal birefringence pattern returned. Reduction in spindle length could be temporally coupled with the reappearance of kinetochores and the reassociation of microtubules with these structures. In contrast to treated and released metaphase cells, anaphase spindles did not return to pretreatment lengths. Replacement of sucrose with medium showed the resumption of chromosome-to-pole motion within 2 min of sucrose removal. Chromosome motion could be correlated with the reappearance of kinetochores and kinetochore microtubules. These data have led us to postulate the existence of two microtubule continuums in the spindle and to discuss their roles in spindle organization and chromosome motion.

Electron acceptance at photosystem II in an oxygen-evolving photosystem II preparation: clear discrimination of two sites of electron acceptance for quinones and quinonediimines associated with a photosystem II preparation.

Oxygen-producing electron transport reactions of a photosystem II preparation are described. The preparation has six major peptides with apparent molecular weights of 36,000, 31,000, 28,000, 27,000, 25,000, and 21,000. Sucrose density gradient centrifugation indicates that the preparation is more homogeneous and more dense than control thylakoid membranes. The preparation photoreduces a number of known photosystem II oxidants including the Class I acceptor, ferricyanide; the Class II acceptor, 2,6-dichloroindophenol; and the Class III acceptor, 2,6-dichlorobenzoquinone. However, quinonediimines such as phenylenediimine are not reduced, suggesting that these substances are reduced at sites in the thylakoid membrane which are not found in the photosystem II preparation. All the oxygen-producing reactions are sensitive to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

Proton involvement with the light-induced hindrance of spin label motion in the lumen of spinach thylakoids.

The light-induced hindrance of spin label motion increases linearly with light intensity. However, it has not been possible to unambiguously demonstrate light saturation due to the very high rates of spin label reduction at high light intensity. The light-induced hindrance of spin label motion may be mimicked in the dark by subjecting thylakoids to appropriately low pH regimes. Uncouplers such as gramicidin-D and methylamine reduce the light-induced hindrance to dark levels as does ethylenedinitrilotetraacetate (EDTA) treatment. Valinomycin plus KCl which destroys the electric potential is only partially effective in reducing the light-induced hindrance. These results indicate that protons in the aqueous lumen of the thylakoids are closely involved with the observed light-induced hindrance of spin label motion.

Chromium oxalate: a new spin label broadening agent for use with thylakoids.

Potassium tris(oxalato)chromate(III) trihydrate (chromium ixalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.

Pathways of silicomolybdate photoreduction and associated photophosphorylation in tobacco chloroplasts.

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.

Concentration-dependent effects of salicylaldoxime on chloroplast reactions.

Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (less than 10 mM), salicyladoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (less than 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.

Mechanism of KCN inhibition of photosystem I.

Experiments with chloroplasts and purified spinach plastocyanin suggest a mechanism for KCN inhibition of Photosystem I. KCN inhibition can be bypassed by a detergent or reversed by replacement of the inactive plastocyanin. KCN bleaches and inactivates purified plastocyanin. KCN releases copper from chloroplast membranes and from purified plastocyanin. Cyanide does not bind to the apoprotein produced when plastocyanin is treated with KCN, and KCN-produced apoplastocyanin has a N-ethylmaleimide-reactive sulfhydryl group not found in holoplastocyanin. Apoplastocyanin is not active in restoring Photosystem I activity to plastocyanin-depleted membranes. Holoplastocyanin restores Photosystem I activities to plastocyanin-depleted membranes prepared from either control or KCN-treated chloroplasts to about the same extent. KCN-treated chloroplast membranes are found to have higher amounts of apoplastocyanin than do control chloroplast membranes. These results offer evidence that KCN removes the copper from plastocyanin in the chloroplast membrane, leaving the inactive apoplastocyanin which is unable to transfer electrons to Photosystem I.

Nuclear magnetic resonance studies of the copper binding sites of blue copper proteins: oxidized, reduced, and apoplastocyanin.

Proton nuclear resonance spectra at 250 MHz of plastocyanins from spinach (Spinacia oleracea) and a blue green alga (Anabaena variabilis) are reported. Spectra of the reduced plastocyanins contain well-resolved peaks from slowly exchangeable N-H, histidine C2-H tyrosine ring, peptide alpha-CH, and high-field protons. The widths of these peaks indicate that the plastocyanins are monomeric. When the plastocyanins are oxidized, several changes in the spectra are observed including disappearance of peaks assigned to two histidine side chains. The pKa' values of the two histidine residues of reduced spinach plastocyanin are abnormally low (4.9 and less than 4.5). These pKa' values become more normal in apoplastocyanin or plastocyanin inhibited by cyanide. The results suggest that the imidazole groups of the two histidine residues are liganded directly to the copper in plastocyanin. The displacement of copper by cyanide is reversed at low pH. Spectra of apo- and reduced plastocyanins show only minor differences. However, the slowly exchangeable protons of plastocyanin exchange more rapidly in the apoprotein. Copper binding apparently does not cause a major reorganization of the protein structure, but the presence of copper does stabilize this structure.