Spontaneous assembly of photosynthetic supramolecular complexes as mediated by the intrinsically unstructured protein CP12

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-GAPDH)-(CP12-2)(2) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-GAPDH)-(CP12-2)(2)-(PRK))(2). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAPDH and PRK during darkness in photosynthetic tissues.     

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information

Background  

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.     

The analysis of the different functions of starch‐phosphorylating enzymes during the development of Arabidopsis thaliana plants discloses an unexpected role for the cytosolic isoform GWD2

The genome of Arabidopsis thaliana encodes three glucan, water dikinases. Glucan, water dikinase 1 (GWD1; EC 2.7.9.4) and phosphoglucan, water dikinase (PWD; EC 2.7.9.5) are chloroplastic enzymes, while glucan, water dikinase 2 (GWD2) is cytosolic. Both GWDs and PWD catalyze the addition of phosphate groups to amylopectin chains at the surface of starch granules, changing its physicochemical properties. As a result, GWD1 and PWD have a positive effect on transitory starch degradation at night. Because of its cytosolic localization, GWD2 does not have the same effect. Single T‐DNA mutants of either GWD1 or PWD or GWD2 have been analyzed during the entire life cycle of A. thaliana. We report that the three dikinases are all important for proper seed development. Seeds from gwd2 mutants are shrunken, with the epidermal cells of the seed coat irregularly shaped. Moreover, gwd2 seeds contain a lower lipid to protein ratio and are impaired in germination. Similar seed phenotypes were observed in pwd and gwd1 mutants, except for the normal morphology of epidermal cells in gwd1 seed coats. The gwd1, pwd and gwd2 mutants were also very similar in growth and flowering time when grown under continuous light and all three behaved differently from wild‐type plants. Besides pinpointing a novel role of GWD2 and PWD in seed development, this analysis suggests that the phenotypic features of the dikinase mutants in A. thaliana cannot be explained solely in terms of defects in leaf starch degradation at night.     

A new combination of methods for the localization,identification, and three-dimensional reconstruction of the sensory endings of articular afferents characterized by electrophysiology

A combination of methods is described to identify and reconstruct corpuscular and non-corpuscular sensory endings of group II and group III nerve fibers following functional examination by electrophysiology. Afferent units activated by electrical stimulation of the medial articular nerve of the cat's knee were analyzed by single fiber recordings and characterized by their responsiveness to mechanical stimuli. The receptive fields of the units were closely demarcated by fine needles when the responses elicited by insertion of the needles were being recorded. After fixation, the tissue around the demarcated field was dissected and histologically processed. Series of semithin sections were cut from the embedded tissue blocks containing the receptive fields. Corpuscular endings of group II fibers and peripheral myelinated group III nerve fibers, presumably corresponding to the characterized units, were identified by light microscopy of semithin sections and localized within the demarcated area. Non-corpuscular endings were identified by electron microscopy of ultrathin sections cut in alternation with, or after re-embedding of, semithin sections. Morphometric analysis of ultrathin section series allowed the measurement of parameters such as the mean axon diameter and the organelle content of the sensory endings. The methods described are appropriate for collecting data that correlate the structural and functional characteristics of sensory endings in deep tissues.     

Effects of four different single exercise sessions on lipids, lipoproteins, and lipoprotein lipase

The purpose ofthis study was to determine the threshold of exercise energyexpenditure necessary to change blood lipid and lipoproteinconcentrations and lipoprotein lipase activity (LPLA) in healthy,trained men. On different days, 11 men (age, 26.7 ± 6.1 yr; bodyfat, 11.0 ± 1.5%) completed four separate, randomly assigned,submaximal treadmill sessions at 70% maximalO₂ consumption. During eachsession 800, 1,100, 1,300, or 1,500 kcal were expended. Compared withimmediately before exercise, high-density lipoprotein cholesterol(HDL-C) concentration was significantly elevated 24 h after exercise(P < 0.05) in the 1,100-, 1,300-, and 1,500-kcal sessions. HDL-C concentration was also elevated(P < 0.05) immediately after and 48 h after exercise in the 1,500-kcal session. Compared with values 24 hbefore exercise, LPLA wassignificantly greater (P < 0.05) 24 h after exercise in the 1,100-, 1,300-, and 1,500-kcal sessions andremained elevated 48 h after exercise in the 1,500-kcal session. Thesedata indicate that, in healthy, trained men, 1,100 kcal of energyexpenditure are necessary to elicit increased HDL-C concentrations.These HDL-C changes coincided with increased LPLA.

     

NAD(P)H:(Quinone-Acceptor) Oxidoreductase of Tobacco Leaves Is a Flavin Mononucleotide-Containing Flavoenzyme

The soluble NAD(P)H:(quinone-acceptor) oxidoreductase [NAD(P)H-QR, EC 1.6.99.2] of Nicotiana tabacum L. leaves and roots has been purified. NAD(P)H-QR contains noncovalently bound flavin mononucleotide. Pairs of subunits of 21.4 kD are linked together by disulfide bridges, but the active enzyme is a homotetramer of 94 to 100 kD showing an isoelectric point of 5.1. NAD(P)H-QR is a B-stereospecific dehydrogenase. NADH and NADPH are electron donors of similar efficiency with Kcat:Km ratios (with duroquinone) of 6.2 x 107 and 8.0 x 107 m-1 s-1, respectively. Hydrophilic quinones are good electron acceptors, although ferricyanide and dichlorophenolindophenol are also reduced. The quinones are converted to hydroquinones by an obligatory two-electron transfer. No spectral evidence for a flavin semiquinone was detected following anaerobic photoreduction. Cibacron blue and 7-iodo-acridone-4-carboxylic acid are inhibitory. Tobacco NAD(P)H-QR resembles animal DT-diaphorase in some respects (identical reaction mechanism with a two-electron transfer to quinones, unusually high catalytic capability, and donor and acceptor substrate specificity), but it differs from DT-diaphorase in molecular structure, flavin cofactor, stereospecificity, and sensitivity to inhibitors. As in the case with DT-diaphorase in animals, the main NAD(P)H-QR function in plant cells may be the reduction of quinones to quinols, which prevents the production of semiquinones and oxygen radicals. The enzyme appears to belong to a widespread group of plant and fungal flavoproteins found in different cell compartments that are able to reduce quinones.     

Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.     

Bony ponticles of the atlas (C1) over the groove for the vertebral artery in humans and primates: polymorphism and evolutionary trends

The aim of this study was to ascertain the distribution in primates of the three possible bony ponticles over the groove for the vertebral artery (ventral, lateral, and dorsal ponticles), in order to attempt to understand the variants observed in humans and to ascertain possible evolutionary trends in primates. The material consisted of 393 atlases of extant nonhuman primates representative of 41 genera, and of 500 human atlases (dried bones of adults). For each atlas, we studied the existence and morphology of the ponticles, and the type of association of these three ponticles on a given side, which are theoretically of eight in number (types A-H). The occurrence of these ponticles varied from complete absence to constant presence, according to the genera and taxa of primates. The presence of each of these ponticles in primates can be interpreted as a primitive or plesiomorphic character, and their absence as a derived or apomorphic character. The strepsirhines-platyrrhines-cercopithecines group, presenting a predominant primitive pattern (type A), appeared to be separated from the colobines-hominoids group, presenting predominant derived patterns (type C in colobines, Pongo pygmaeus, and Pan troglodytes, and the more derived type D in Hylobates, Gorilla gorilla, and Homo sapiens). The last derived stage, corresponding to the disappearance of the three atlantal ponticles (type H), was only observed in some individuals in hominoids. A marked intraspecific polymorphism characterized the hominoids. The presence of lateral and dorsal ponticles in humans appeared to correspond to their persistence within the progressive disappearance of the atlantal ponticles, constituting an evolutionary tendency characteristic of primates and particularly of hominoid evolution.