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J Barber

Publications and source records attributed to J Barber.

At least 127 records · Page 7Linked to original sources
PubMed1994

An aroA homologue from Synechocystis sp. PCC 6803.

In this study, we report the entire nucleotide sequence of an aroA homologue encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), isolated from the cyanobacterium Synechocystis sp. PCC 6803. The proposed coding region is an open reading frame of 447 amino acids. The deduced sequence of the gene product is particularly similar to the Gram+ EPSPS sequences available to date, in particular to that in Bacillus subtilis. Analysis of the Synechocystis putative EPSPS sequence does not lead to an obvious explanation for the natural tolerance of this cyanobacterium to glyphosate.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1994

Isolated photosynthetic reaction center of photosystem II as a sensitizer for the formation of singlet oxygen. Detection and quantum yield determination using a chemical trapping technique.

Singlet oxygen formation by photosystem II reaction centers isolated from Pisum sativum has been detected by two chemical trapping techniques: histidine-dependent oxygen uptake and bleaching of p-nitrosodimethyl-aniline by the intermediary endoperoxide of histidine. The quantum yield of singlet oxygen formation determined by these methods was estimated to be 0.16 by comparison with the known quantum yields of standard singlet oxygen sensitizers. Singlet oxygen was formed on illumination of reaction centers under conditions that lead to formation of the triplet state of the primary electron donor, P680. Experiments with deuterated buffer and active oxygen scavengers indicated that singlet oxygen was the only active oxygen species produced by this reaction. Neither azide nor histidine, which are scavengers of singlet oxygen, protected against photobleaching of the chlorophyll of reaction centers that occurs concomitantly with singlet oxygen formation, suggesting that bleaching involves singlet oxygen generated within the protein matrix of the complex. Singlet oxygen sensitized exogenously by rose bengal (when excited specifically at 550 nm) was also found to bleach reaction center chlorophyll in a manner similar to the intrinsic mechanism. We conclude that singlet oxygen formed within the hydrophobic interior of the reaction center attacks the chlorophylls of P680, and presumably also amino acids in the vicinity, and that only the singlet oxygen that escapes to the medium is affected by added scavengers or deuterated medium. These experiments extend our earlier report of the detection of singlet oxygen by its luminescence at 1270 nm when isolated photosystem II reaction centers are illuminated (Macpherson, A. N., Telfer, A., Barber, J., and Truscott, T. G. (1993) Biochim. Biophys. Acta 1143, 301-309). Moreover, our results support the hypothesis that production of singlet oxygen underlies the vulnerability of photosystem II to photodamage and hence necessitates the rapid turnover of the D1 protein of the reaction center.

Fabaceae↗
PubMed1994

In vivo and in vitro photoinhibition reactions generate similar degradation fragments of D1 and D2 photosystem-II reaction-centre proteins.

Isolation of photosystem-II reaction centres from pea leaves after photoinhibitory treatment at low temperature (0-1 degrees C) has provided evidence for the mechanism of degradation of the D1 protein in vivo. These isolated reaction centres did not appear to be spectrally distinct from preparations obtained from control leaves that had not been photoinhibited. Breakdown fragments of both the D1 and D2 proteins were, however, found in preparations isolated from photoinhibited leaves, and showed similarities with those detected when isolated reaction centres were exposed to acceptor-side photoinhibition. Analyses of the origin of D1 fragments indicated that the primary cleavage site of this protein was between transmembrane helices IV and V indicative of the acceptor-side mechanism for photoinhibition. The origins of other D1 protein fragments indicate that some donor-side photoinhibition may also have occurred in vivo under the conditions employed. We have shown that the spectral and functional integrating of the isolated photosystem II reaction centre complex is resistant to proteolytic cleavage by trypsin. Use of a more non-specific protease (subtilisin), however, caused significant destabilisation of the special pair of chlorophylls constituting the primary electron donor, P680, with a consequential loss of functional activity. Thus, it is possible that specific cleavage of photosystem-II reaction-centre proteins may occur in vivo following photoinhibitory damage without a significant change in structural integrity, a conclusion supported by the finding that photodamaged and normal reaction centres were isolated together.

Fabaceae↗
PubMed1994

Degradation of D2 protein due to UV-B irradiation of the reaction centre of photosystem II.

Exposure of isolated reaction centres of photosystem II to UV-B radiation generates specific breakdown products of the D2 protein. When the quinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone is present a 22 kDa fragment containing the N-terminus of the mature protein is generated. Concomitant with the appearance of the N-terminal fragment, two fragments containing the C-terminus of the D2 protein having apparent molecular masses around 10-12 kDa are observed. It is concluded that the primary cleavage occurs in the hydrophilic loop linking putative transmembrane segments IV and V. No such cleavage was observed when silicomolybdate was used as an electron acceptor, suggesting that this UV-B damage is dependent on binding of the added quinone to the QA site.

Benzoquinones↗
PubMed1994

Transduction of human melanoma cells with interleukin-2 gene reduces tumorigenicity and enhances host antitumor immunity: a nude mouse model.

Human melanoma tumor cells were genetically modified in vitro by transferring the interleukin-2 (IL-2) gene via a retroviral vector into established or fresh tumor cells. In addition, human melanoma cells were transduced in vivo by the direct injection of the IL-2/retroviral vector into melanoma xenografts in nude mice. The gene-modified melanoma cells expressed the IL-2 cytokine gene and secreted biologically active IL-2. Transduction of melanoma cells with the IL-2 gene did not affect the antigenic profile of the cells, but caused a strong abrogation of their tumorigenicity. One million parental cells formed subcutaneous tumors in nude mice. In contrast, various doses of up to 20 x 10(6) IL-2-transduced cells failed to form tumor in the mice. Coinjection of IL-2-producing cells with parental cells inhibited tumor formation even when highly tumorigenic doses of parental cells were used. Histochemical analysis of the injection sites of IL-2-modified cells showed an influx of host immune cells, predominantly macrophages, as early as the third day after inoculation. Neutrophils, mast cells, and eosinophils were also seen in the inflammatory exudate. Eventually, transduced cells showed signs of degeneration and necrosis and ultimately died in 4 weeks. Macrophages were seen in parental tumor sites only during the first few days after injection, and then parental tumors exhibited fast, progressive growth. The study suggests that melanoma cells transduced with the IL-2 cytokine gene may provide an effective vaccine for melanoma patients, whereas the in vivo transduction of tumors with cytokine genes is feasible and may represent a novel approach for the immunotherapy of cancer patients.

Animals↗
PubMed1994

Neospora caninum in dogs: detection of antibodies by ELISA using an iscom antigen.

An indirect enzyme linked immunsorbent assay (ELISA) for detection of antibodies to Neospora caninum in serum from dogs is described. Extracted tachyzoite proteins incorporated into immunostimulating complexes (iscoms) were used as coating antigen. A mixture of a monoclonal antibody to dog immunoglobulin G and a horse radish peroxidase conjugated antibody to mouse Ig was used to detect bound antibody. When the iscom preparation was analysed by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis it appeared to consist of a restricted number of proteins compared with whole parasite homogenates. In immunoblot analysis, using N. caninum positive sera from rabbits and dogs as probes, the major antigens recognized had approximate molecular weights between 30 and 45 and 17 to 19 kDa. Compared with an ELISA using a crude solubilized tachyzoite antigen, the iscom ELISA substantially improved the sensitivity and specificity (to 97.6% and 95.6%, respectively, against an immunofluorescence test, IFAT, as indicator of true status). There was a statistically significant positive correlation between IFAT titres and iscom ELISA OD450 values. The iscom ELISA absorbances (and the IFAT titres) of dogs with proven clinical infections were not higher than those from nonclinically affected, putatively infected dogs.

Animals↗
PubMed1994

Disaster preparedness. Ready for earth, air, fire and water (and who knows what else)?

Already the 1990s might be described as a decade of disasters: From the devastation of hurricanes on the East Coast to the havoc of floods in the Midwest to the destruction of earthquakes and civil unrest on the West Coast, the past few years clearly show that disasters can strike anywhere--and at any time. For health facilities caught in the middle of these crises, emergency preparedness means much more than just meeting the basic requirements of the Joint Commission on Accreditation of Healthcare Organizations or complying with state and local regulations. Rather, for most health facilities, preparing for disasters is an integral part of ensuring that they can continue to provide adequate, high-quality patient care under almost any circumstances. This report, the first in a series of articles, examines how health facility managers coped once they found themselves in the midst of major disasters, what lessons they learned about emergency preparedness as a result of their experiences, and what others can learn from these difficult situations.

California↗
PubMed1993

A functional model for the role of cytochrome b559 in the protection against donor and acceptor side photoinhibition.

A quinone-independent photoreduction of the low potential form of cytochrome b559 has been studied using isolated reaction centers of photosystem II. Under anaerobic conditions, the cytochrome can be fully reduced by exposure to strong illumination without the addition of any redox mediators. Under high light conditions, the extent and rate of the reduction is unaffected by addition of the exogenous electron donor Mn2+ and, during this process, no irreversible damage occurs to the reaction center. However, prolonged illumination in strong light brings about irreversible bleaching of chlorophyll, indicative of photoinhibitory damage. When the cytochrome is fully reduced and excess Mn2+ is present, the effect of moderate light is to facilitate the photoaccumulation of reduced pheophytin. The dark reoxidation of the reduced cytochrome is very slow under anaerobic conditions but significantly speeded up on addition of oxidized 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. From these results it is suggested that the low potential form of cytochrome b559 can accept electrons directly from reduced pheophytin and in so doing help to protect the reaction center against acceptor side photoinhibition as suggested by Nedbal et al. [Nedbal, J., Samson, G. & Whitmarsh, J. (1992) Proc. Natl. Acad. Sci. USA 89, 7929-7933]. This conclusion has been incorporated into a model that further suggests that in its high potential form the cytochrome primarily acts to protect against donor side photoinhibition due to increased lifetime of highly oxidized species as previously proposed by Thompson and Brudvig [Thompson, L. & Brudvig, G. W. (1988) Biochemistry 27, 6653-6658]. The particular feature of our scheme is that it incorporates reversible interconversion between the two redox forms so as to protect against either type of photoinhibition.

Cytochrome b Group↗
PubMed1993

Rate of oxidation of P680 in isolated photosystem 2 reaction centers monitored by loss of chlorophyll stimulated emission.

We have continued our studies of the primary photochemistry of isolated photosystem 2 reaction centers using femtosecond transient absorption spectroscopy. Experiments were performed over a wide range of excitation and probe wavelengths, using several data collection time scales. This has enabled us to resolve five different lifetimes ranging between 100 fs and 200 ps plus a nanosecond component. We demonstrate here and elsewhere [e.g., Durrant, J.R., Hastings, G., Joseph, D. M., Barber, J., Porter, G., & Klug, D. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11632-11636] that the kinetic spectra associated with all but two of these lifetimes are clearly distinguishable. We have previously reported that a 21-ps lifetime is associated with pheophytin reduction [Hastings, G., Durrant, J. R., Hong, Q., Barber, J., Porter, G., & Klug, D. R. (1992) Biochemistry 31, 7638-7647]. In this paper, we show that it is possible to spectrally and temporally resolve stimulated emission from PS2 reaction centers with great accuracy and that this stimulated emission is largely unaffected by those kinetic components which are faster than 21 ps. The observation of a distinct stimulated emission band allows us to distinguish charge-separated states from chlorin singlet states. In this way, we are able to show that the proportion of charge-separated states prior to the 21-ps component is between 0% and 25%. We also show that the shape of the spectrum which we obtain for the state P680+Ph- is essentially invariant between 100 ps and 9 ns, and is the same as that previously reported for P680+Ph- by other researchers.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll↗
PubMed1993

Acceptor side mechanism of photoinduced proteolysis of the D1 protein in photosystem II reaction centers.

A 23-kDa breakdown product, containing the N terminus of the D1 protein, has been detected after photoinhibitory treatment of isolated photosystem II (PSII) reaction centers. The ability to induce charge separation in the reaction center and the presence of oxygen seem to be required for the generation of this fragment. It is suggested that, under these conditions, the initial light-induced damage to the complex occurs via singlet oxygen generated by the P680 triplet state and contrasts with the situation when an electron acceptor is present and donor-side photoinhibition gives rise to a 24-kDa C-terminal fragment of the D1 protein. The temperature sensitivity of the appearance of the 23-kDa N-terminal fragment suggests that the cleavage is not by a direct photochemical process but that it is proteolytic in nature, being triggered possibly by a conformational change induced by singlet oxygen-mediated photodestruction of the P680 chlorophylls. The existence of an intrinsic serine-type protease, within the reaction center itself, is supported by inhibition of the appearance of the 23-kDa N-terminal fragment by stoichiometric levels of soybean trypsin inhibitor. It seems likely that the 23-kDa N-terminal fragment which we have detected is the same as that identified in vivo by Greenberg et al. [Greenberg, B. M., Gaba, V., Mattoo, A. K., & Edelman, M. (1987) EMBO J. 6, 2865-2869] and originates from the acceptor-side mechanism advocated by Vass et al. [Vass, I., Styring, S., Hundal, T., Koivuniemi, A., Aro, E.-M., & Andersson, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1408-1412].

Bacterial Proteins↗
PubMed1993

The genes aroA and trnQ are located upstream of psbO in the chromosome of Synechocystis 6803.

We have identified the existence of two genes, trnQ and aroA, located upstream of the psbO gene in Synechocystis sp. PCC 6803. The trnQ gene encodes a glutamine-specific transfer RNA (tRNA(Gln)) and the sequence given is the first reported for any cyanobacterium. The gene seems to exist as a single copy since its deletion results in non-viable mutation. The aroA gene encodes for 5-enolpyruvylshikimate 3-phosphate synthase and its discovery in the genome of Synechocystis 6803 is the first genetic evidence for the existence of the shikimate biosynthetic pathway in cyanobacteria. Interestingly, the partial sequence shares close homologies with the sequences of aroA from Gram-positive bacteria.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1993

Detection of a 10 kDa breakdown product containing the C-terminus of the D1-protein in photoinhibited wheat leaves suggests an acceptor side mechanism.

Photoinhibition of intact leaves of wheat generates a 10 kDa breakdown product which is clearly observed both at 4 degrees C and 25 degrees C. Selective immunoblotting has shown that the 10 kDa fragment contains the C-terminus of the D1-protein and, under the conditions employed, supports an acceptor side mechanism for photoinhibition in vivo. Although a corresponding 23 kDa N-terminal D1-fragment was not detected our results are consistent with the argument that the primary cleavage site is in the loop joining putative transmembrane segments IV and V.

Bacterial Proteins↗