Stabilisation of PS-II-mediated electron transport in oxygen-evolving PS II core preparations by the addition of compatible co-solutes.

Addition of high concentrations of compatible co-solutes such as sugars, sugar alcohols and polyols has recently been shown to lead to marked increases in the thermal stability of oxygen-evolution in chloroplasts (Williams et al. (1992) Biochim. Biophys. Acta 1099, 137-144). In this paper, a similar stabilisation is demonstrated for oxygen-evolving PS II core preparations. The presence of such co-solutes appears, however, to have no ability to stabilise PS II reaction-centre preparations against heat-induced changes in their absorption spectrum. Nor do they protect electron transport from artificial electron donors in PS II core preparations lacking the extrinsic 33 kDa polypeptide of the oxygen-evolution system. Measurements performed on core preparations retaining the 33 kDa polypeptide but lacking the 17 kDa and 23 kDa polypeptides indicate that the co-solutes protect PS-II-mediated electron transport by stabilising the binding of the 33 kDa polypeptide to the core complexes. These findings are discussed in terms of an extension of the general principles underlying the Hofmeister effect observed for soluble proteins to the stabilisation of photosynthetic membrane preparations.

Evidence that pyrene excimer formation in membranes is not diffusion-controlled.

Kinetic and steady-state measurements of pyrene fluorescence in a variety of model membranes are evaluated in terms of the theory of collisional excimer formation. In the region of 10(-3)-0.1 M pyrene, molecular fluorescence decay in membranes is biphasic and the two component lifetimes do not depend on the pyrene concentration. The lifetime data are consistent with the rate constant for collisional excimer formation being of the order 10(6) M-1 X s-1 or less. The concentration dependence of the component amplitudes is inconsistent with the theory of collisional excimer formation and suggests that pyrene exists in two forms in membranes: a slowly diffusing monomeric form and an aggregated form. The component of molecular fluorescence decay associated with aggregated pyrene is highly correlated with steady-state excimer fluorescence, suggesting that excimer fluorescence in membranes arises from aggregated pyrene in which excimers are formed by a static rather than a collisional mechanism. It is suggested that the concentration dependence of excimer to molecular fluorescence intensity ratios in membranes is related to the equilibrium constant for exchange between monomeric and aggregated pyrene forms rather than to the collisional excimer formation rate constant.

Structural organisation and lipid composition of the epicuticular accessory layer of infective larvae of Trichinella spiralis.

The epicuticle of infective larvae of Trichinella spiralis represents the interface between this intracellular nematode parasite and the cytosol of mammalian skeletal muscle. The macromolecular structures that make up the epicuticle were studied by freeze-fracture electron microscopy and compositional analysis. Three fracture planes were observed: one with a typical plasma membrane-type bilayer organisation which was overlaid by two extended layers of lipid in an inverted cylindrical configuration. This overall structure remained unchanged in response to variations in temperature between 20 degrees C and 45 degrees C. The lipid cylinders were on average 6.8 nm in diameter, with randomly-associated particles that were not dissociated by high-salt treatment, indicative of hydrophobically associated proteins. The majority of the lipids were non-polar, consisting of cholesterol, cholesterol esters, mono- and tri-glycerides, and free fatty acids. Three major classes of phospholipids were identified: phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine. Total lipid extracts did not adopt an inverted cylindrical or micellar configuration on isolation, but formed flat sheets of lamellae as did the purified polar and non-polar fractions of the lipids. Isolated lipids did not undergo thermally-induced polymorphism between 20 degrees C and 60 degrees C and there was no pH dependency of the structures adopted. The fatty acid saturation levels of the phospholipids were compatible with the observation that they did not form polymorphic structures on isolation. We suggest that this unusual configuration is probably stabilised by the associated (glyco)proteins and may be required for selective permeation of nutrients from the host cell cytosol and/or for maintaining the high curvature of the parasite within the cell.

Identification of the psbH gene product as a 6 kDa phosphoprotein in the cyanobacterium Synechocystis 6803.

The product of the psbH gene has been identified in Synechocystis 6803 thylakoid membranes as a 6 kDa phosphoprotein. This protein becomes phosphorylated in vitro despite the fact that in cyanobacteria it is truncated at the N-terminus such that the phosphorylation site identified in the higher plant protein is missing. Phosphorylation occurred both in the light and in the dark but was inhibited by oxidising conditions, DCMU and zinc ions. The cyanobacterial 6 kDa phosphoprotein degrades when the membranes are subjected to high intensity illumination.

A reversible protein phosphorylation system is present at the surface of infective larvae of the parasitic nematode Trichinella spiralis.

Trichinella spiralis infective larvae have externally oriented enzymes catalysing reversible protein phosphorylation on their surface. Incubation of larvae with exogenous ATP resulted in phosphorylation of surface bound and released proteins. Exposure of the parasites to bile, a treatment which renders them infective for intestinal epithelia, resulted in increased release of protein and an altered profile of phosphorylation. Both serine/threonine and tyrosine phosphorylation and dephosphorylation reactions took place at the parasite surface. Examination of the structural characteristics of the larvae following exposure to bile showed that the non-bilayer surface coat was not shed but was structurally reorganised.

Comparison of the D1/D2/cytochrome b559 reaction centre complex of photosystem two isolated by two different methods.

Photosystem 2 reaction centre complexes prepared either by solubilisation with Triton X-100 and subsequent exchange into dodecyl maltoside or by a procedure involving a combination of dodecyl maltoside and LiClO4, were characterised in terms of chlorophyll a, pheophytin a, beta-carotene and cytochrome b559 content. Time-resolved chlorophyll fluorescence decay kinetics were measured using both types of complexes. Our data show that the isolated photosystem two reaction centre complex contain, for two pheophytin a molecules, close to six chlorophyll a, two beta-carotene and one cytochrome b559. No major differences were observed in the composition or the kinetic characteristics measured in the samples prepared by the different procedures. Time-resolved fluorescence measurements indicate that more than 94% of the chlorophyll a in both preparations is coupled to the reaction centre complex.

Identification and composition of lipid classes in surface and somatic preparations of adult Brugia malayi.

The cuticle of adult Brugia malayi is the organisms's major point of interaction with the mammalian host environment. We therefore undertook an investigation in order to define the lipid composition of this outermost layer of the parasite. The lipid class and fatty acid composition of the cuticle of adult Brugia malayi was examined by surface specific radioiodination, organic extraction, thin layer chromatography and gas chromatography. The data were compared with those derived from similar analyses of somatic preparations of the parasites. The composition of the cuticular lipid fraction was found to be highly unusual and distinct from that of the internal lipids. Cholesterol esters and wax esters were absent from the cuticular lipid fraction, which was however enriched in unesterified fatty acids. The major polar lipids in both cuticular and somatic preparations were phosphatidylcholine and phosphatidylethanolamine, but unusually high levels of lysophosphatidylethanolamine were observed in the cuticular extracts. Analyses of cuticular polar lipids indicated that there is an asymmetric distribution of the fatty acids in phosphatidylethanolamine, assuming that lysophosphatidylethanolamine is derived from deacylation of the former molecule in the cuticle. The major fatty acids in all lipid fractions examined were the 18-carbon, mono- and di-unsaturated type, while significant amounts of palmitic, palmitoleic, stearic and eicosatrienoic acids were also found. A highly unusual feature of the cuticular lipid fraction was that it contained large amounts of a novel polar lipid species which, on exposure to atmospheric oxygen, degraded to a hydrophobic and a hydrophilic moiety. This polar lipid was absent from the somatic preparations. The data are discussed in terms of the possible resistance or susceptibility of the parasite to reactive oxygen species.

Identification of serine/threonine protein kinases secreted by Trichinella spiralis infective larvae.

Serine/threonine protein kinase activity was identified in excretory/secretory (ES) products of Trichinella spiralis infective larvae, via phosphorylation of exogenous and endogenous substrates. Protein kinase activity was identified as an authentic secretory product via blockade of release into culture medium by brefeldin A. Enzyme activity was reductant-dependent, and the relative resistance to a panel of inhibitors suggested that it could not be readily assigned to any of the major documented subfamilies of serine/threonine protein kinases. There was no evidence for protein tyrosine kinase activity in ES products. The major phosphorylated proteins in this compartment resolved at 50 and 55 kDa by SDS-PAGE, and are therefore designated pp50/55. These proteins contained mainly phosphoserine, and appear to represent differentially glycosylated variants of a 35 kDa polypeptide, modified via the addition of three and four N-linked oligosaccharides, respectively. An autophosphorylation assay following separation by SDS-PAGE identified two protein kinases of 70 and 135 kDa in ES products.

Resistance of filarial nematode parasites to oxidative stress.

All filariae examined to date express a comprehensive repertoire of both cytoplasmic and secreted anti-oxidant enzymes, although significant differences exist between species and life-cycle stages. Adult Brugia malayi, Dirofilaria immitis and Onchocerca volvulus secrete CuZn superoxide dismutases, and the former two species also secrete a selenocysteine-independent glutathione peroxidase. This enzyme has been localised to the cuticular matrix of B. malayi, and the preferential reduction of fatty acid- and phospholipid hydroperoxides suggests that it may protect cuticular membranes from oxidative damage rather than directly metabolise hydrogen peroxide. Adult O. volvulus may compensate for an apparent deficiency in expression of this enzyme via a secreted variant of glutathione S-transferase. Recent studies have identified a highly expressed family of enzymes collectively termed peroxiredoxins, which most probably play an essential role in reduction of hydroperoxides. Data from cDNA cloning exercises indicate that all filarial species examined thus far express at least two peroxiredoxin variants which have been localised to diverse tissues. In-vitro studies have shown that B. malayi are particularly resistant to oxidative stress, and that the parasites do not rely solely on enzymatic mechanisms of defence. Cuticular lipids are relatively resistant to lipid peroxidation due to the low unsaturation indices of the constituent fatty acyl residues, but complete protection is afforded by the presence of alpha-tocopherol, presumably assimilated from host extracellular fluids. Brugia malayi are also relatively resistant to nitric oxide-mediated toxicity, and this may be due in part to incomplete dependence on aerobic metabolism. Little is known of potential mechanisms for detoxification of nitric oxide derivatives and adaptive responses to oxidative stress in general, and these represent goals for future research.

Mutation spectrum and phenotypic manifestation in FSHD Greek patients.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic myopathy with a remarkable intra- and inter-familial clinical heterogeneity. This study reports the clinical and genetic analysis of 133 individuals from 71 unrelated Greek families based on a revised clinical severity score (rCSS) index which was developed for clinical assessment regarding the disease progression. A high ratio (31/62, 50%) of probands' family members was found to be asymptomatic or minimally affected gene carriers of a contracted 4q allele. Moreover, a notable clinical variability of FSHD is reported concerning the detection of an identical de novo 13 b EcoRI fragment in monozygotic twins, as well as indications of founder effect. This is the first survey that presents data of FSHD families from an East Mediterranean country supporting the speculation that the prevalence of disease might be significantly underestimated and that synergistic factors could play an essential role on the progression of the disease.

What role does sulpholipid play within the thylakoid membrane?

Sulphoquinovosyldiacylglycerol is a negatively charged lipid which exists in the thylakoid membrane. It is proposed that a large proportion of this acidic lipid does not form a part of the bulk lipid matrix but is closely associated with protein complexes where it is tightly bound and participates in either optimising catalytic activities, or maintaining the complexes in a functional conformation. Experimental evidence for this proposal is emerging from studies with isolated photosystem 2, and coupling factor complexes.

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina.

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using (32)P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.

Secreted variant of nucleoside diphosphate kinase from the intracellular parasitic nematode Trichinella spiralis.

The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts gamma-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.

Brugia malayi: ultrastructural morphology of the cuticular surface membranes of adult parasites.

We have obtained and examined several fracture planes close to the surface of the cuticle of adult Brugia malayi by freeze-fracture electron microscopy. We observed three exocytoplasmic and two protoplasmic fracture faces exhibiting the periodic annulations characteristic of the outer layers of the cuticle. The outermost exocytoplasmic fracture face we observed contained small randomly distributed particles and appears to originate from within the epicuticle. The second exocytoplasmic face appeared as a featureless surface representing a nonmembranous proteinaceous structural arrangement adjacent to the epicuticle. The innermost fracture plane was represented by an exocytoplasmic face with a morphology dominated by large particles regularly appearing as organised linear arrays. This fracture plane seems to originate from a membrane-like organisation directly below the annulated proteinaceous layer. The two annulated protoplasmic faces were exposed by two discrete planes of fracture and were complementary to the exocytoplasmic planes. The above observations are considered in terms of the overall structural organisation of the cuticle of adult B. malayi.

Characterization of a platelet-activating factor acetylhydrolase secreted by the nematode parasite Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis, a small nematode parasite of the gastrointestinal tract of rodents, secretes an enzyme that cleaves the proinflammatory molecule platelet-activating factor to its inactive lyso-form. The enzyme activity of Ca(2+)-dependent and does not exhibit interfacial activation. It does not require the addition of reducing agents for maximal activity, and is not inhibited by thiol-active reagents. Sensitivity of inhibitors suggests the involvement of serine and histidine residues in the enzyme activity. As described for other platelet-activating factor acetylhydrolases, it cannot cleave, nor is it inhibited by, long-chain diacyl phospholipids that are typical substrates for phospholipases A2. The purified enzyme was resolved by SDS/PAGE as a heterodimer composed of two protein subunits with apparent molecular masses of 38 and 25 kDa. The properties of the nematode enzyme thus differ from those described for the mammalian enzymes, but are more closely related to those of an acetylhydrolase than a phospholipase.

Brugia malayi: resistance of cuticular lipids to oxidant-induced damage and detection of alpha-tocopherol in the neutral lipid fraction.

We have examined the susceptibility of cuticular membrane lipids of Brugia malayi to oxidants generated in vitro. Live parasites as well as extracted cuticular lipids were treated with hydrogen peroxide and hypochlorous acid and the extent of lipid peroxidation was quantified. The cuticular membranes of B. malayi were found to be resistant to lipid peroxidation at hydrogen peroxide concentrations which were lethal to the organism. This resistance was partly due to the inherently low unsaturation indices of the fatty acyl residues, but complete protection was afforded by lipid-soluble antioxidants present in the neutral lipid fraction of the parasites. We have identified alpha-tocopherol as a major antioxidant present in both adult and microfilarial B. malayi. In addition, we report that although hypochlorous acid chemically modifies isolated parasite lipids, the latter do not appear to be the primary substrate for the oxidant in live worms. The data are discussed in terms of the susceptibility of B. malayi to products of the respiratory burst from activated myeloid cells.

Heterologous expression and enzymatic properties of a selenium-independent glutathione peroxidase from the parasitic nematode Brugia pahangi.

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).

A method for estimating lateral diffusion coefficients in membranes from steady-state fluorescence quenching studies.

The Stern-Volmer theory, in which the quantum yield ratio (Io/I) depends linearly on the quencher concentration, will typically be inapplicable to fluorescence quenching in membranes. Numerical analysis shows that diffusion-controlled quenching results in a nonlinear concentration dependence for diffusion coefficients less than or of the order of 10(-6) cm2 s-1 and probe fluorescence lifetimes in the region of 10-100 ns. Lateral diffusion coefficients in membranes are typically overestimated an order of magnitude or more by the Stern-Volmer theory. An alternative empirical method is presented, which represents nonlinear concentration curves by a single parameter linear approximation determined by a least-squares analysis. The fitting parameter, P, depends on the interaction distance, the membrane thickness, the maximum extent of quenching and, in the case of biexponential probe fluorescence decay, the fluorescence kinetic parameters. P is presented in tabular form for a useful range of these parameters. The method is used to estimate diffusion coefficients for plastoquinone and plastoquinol from pyrene fluorescence quenching in soya bean phosphatidylcholine liposomes. It is found that the diffusion coefficients are nearly equal and in the region of 1.3-3.5 X 10(-7) cm2 s-1 for interaction radii of 1.5-0.5 nm, respectively.

psbG is not a photosystem two gene but may be an ndh gene.

A gene of the chloroplast genome has been designated the psbG gene on the basis that in maize the gene product is a 24-kDa polypeptide of photosystem two (PS2) (Steinmetz, A. A., Castroviejo, M., Sayre, R. T., and Bogorad, L. (1986) J. Biol. Chem. 261, 2485-2488). We have located and sequenced the equivalent gene in wheat (Triticum aestivum) and have raised specific antibodies to the gene product following its expression in Escherichia coli as a beta-galactosidase fusion protein. Using these antibodies, we have investigated the location of the gene product in various thylakoid membrane fractions of pea (Pisum sativum). The gene product of apparent molecular mass 27-28 kDa is severely depleted in PS2-enriched membrane preparations and its distribution between stromal and granal regions of the membrane is distinct to that of the psbC gene product which is known to be a core polypeptide of PS2. We therefore conclude that psbG does not code for a component of PS2 but instead suggest that it is present in a novel protein complex of the thylakoid membrane. On the basis of 1) the conserved overlap between psbG and ndhC, a chloroplast gene which shows significant homology to a mitochondrial gene that codes for a subunit of the NADH-ubiquinone oxidoreductase of mitochondria, and 2) sequence similarity between the psbG gene product and the ndh gene product of E. coli, which codes for a respiratory NADH dehydrogenase, we propose that this ill-defined complex functions as a NADH or NADPH-plastoquinone oxidoreductase.