Comparative histological studies on properties of polysaccharides secreted by vomeronasal glands of eight Laurasiatheria species.

Most mammalian species have a vomeronasal organ that detects specific chemical substances, such as pheromones. Mucous fluid covering the vomeronasal sensory epithelium is secreted by vomeronasal glands, and the properties of these fluids have been suggested to be involved in chemical detection. Histological studies using periodic acid-Schiff (PAS) and Alcian blue pH 2.5 (AB) stains, which respectively detect natural and acidic polysaccharides, have suggested variations in the nature of the vomeronasal glands among species. Here, we investigated the responsivity of the vomeronasal glands to PAS and AB stains in eight Laurasiatheria species. All species studied herein possessed vomeronasal glands that stained positive for PAS, like other many reported species. The vomeronasal glands of dogs and minks - like rodents, were AB-negative, whereas those of cows, goats, sika deer, musk shrews and two bat species were positive. Considering the present findings and previous reports, the vomeronasal glands in most of Laurasiatheria species appear to be fundamentally abundant in acidic polysaccharides, whereas those in carnivores essentially contains neutral polysaccharides.

Natural discourse reference generation reduces cognitive load in spoken systems.

The generation of referring expressions is a central topic in computational linguistics. Natural referring expressions - both definite references like 'the baseball cap' and pronouns like 'it' - are dependent on discourse context. We examine the practical implications of context-dependent referring expression generation for the design of spoken systems. Currently, not all spoken systems have the goal of generating natural referring expressions. Many researchers believe that the context-dependency of natural referring expressions actually makes systems less usable. Using the dual-task paradigm, we demonstrate that generating natural referring expressions that are dependent on discourse context reduces cognitive load. Somewhat surprisingly, we also demonstrate that practice does not improve cognitive load in systems that generate consistent (context-independent) referring expressions. We discuss practical implications for spoken systems as well as other areas of referring expression generation.

The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity.

Deletion of a region of the human immunodeficiency virus type 2 (HIV-2) 5' leader RNA reduces genomic RNA encapsidation to about 5% that of wild-type virus with no defect in viral protein production but severely limits virus spread in Jurkat T cells, indicating that this region contains a major cis-acting encapsidation signal, or psi (Psi). Being upstream of the major splice donor, it is present on all viral transcripts. We have shown that HIV-2 selects its genomic RNA for encapsidation cotranslationally, rendering wild-type HIV-2 unable to encapsidate vector RNAs in trans. Virus with Psi deleted, however, encapsidates an HIV-2 vector, demonstrating competition for Gag protein. HIV-2 overcomes the lack of packaging signal location specificity by two novel mechanisms, cotranslational packaging and competition for limiting Gag polyprotein.

Redox signalling in chloroplasts and mitochondria: genomic and biochemical evidence for two-component regulatory systems in bioenergetic organelles.

Redox chemistry is central to the primary functions of chloroplasts and mitochondria, that is, to energy conversion in photosynthesis and respiration. However, these bioenergetic organelles always contain very small, specialized genetic systems, relics of their bacterial origin. At huge cost, organellar genomes contain, typically, a mere 0.1% of the genetic information in a eukaryotic cell. There is evidence that chloroplast and mitochondrial genomes encode proteins whose function and biogenesis are particularly tightly governed by electron transfer. We have identified nuclear genes for 'bacterial' histidine sensor kinases and aspartate response regulators that seem to be targeted to chloroplast and mitochondrial membranes. Sequence similarities to cyanobacterial redox signalling components indicate homology and suggest conserved sensory and signalling functions. Two-component redox signalling pathways might be ancient, conserved mechanisms that permit endogenous control over the biogenesis, in situ, of bioenergetic complexes of chloroplasts and mitochondria.

Molecular recognition in thylakoid structure and function.

In photosynthesis, light-harvesting chlorophyll molecules are shunted between photosystems by phosphorylation of the protein to which they are bound. An anchor for the phosphorylated chlorophyll-protein complex has now been identified in the reaction centre of chloroplast photosystem I. This finding supports the idea that molecular recognition, not membrane surface charge, governs the architecture of the chloroplast thylakoid membrane. We describe a model for the chloroplast thylakoid membrane that is consistent with recent structural data that specify the relative dimensions of intrinsic protein complexes and their dispositions within the membrane. Control of molecular recognition accommodates membrane stacking, lateral heterogeneity and regulation of light-harvesting function by means of protein phosphorylation during state transitions--adaptations that compensate for selective excitation of photosystem I or photosystem II. High-resolution structural description of membrane protein-protein interactions is now required to understand thylakoid structure and regulation of photosynthesis.

Bioinformatics and discovery: induction beckons again.

With the flood of information from genomics, proteomics, and microarrays, what we really need now is the computer software to tell us what it all means. Or do we?

Protein tyrosine phosphorylation in the transition to light state 2 of chloroplast thylakoids.

Redox dependent protein phosphorylation in chloroplast thylakoids regulates distribution of excitation energy between the two photosystems of photosynthesis, PS I and PS II. Several thylakoid phosphoproteins are known to be phosphorylated on N-terminal threonine residues exposed to the chloroplast stroma. Phosphorylation of light harvesting complex II (LHC II) on Thr-6 is thought to account for redistribution of light energy from PS II to PS I during the transition to light state 2. Here, we present evidence that a protein tyrosine kinase activity is required for the transition to light state 2. With an immunological approach using antibodies directed specifically towards either phospho-tyrosine or phospho-threonine, we observed that LHC II became phosphorylated on both tyrosine and threonine residues. The specific protein tyrosine kinase inhibitor genistein, at concentrations causing no direct effect on threonine kinase activity, was found to prevent tyrosine phosphorylation of LHC II, the transition to light state 2, and associated threonine phosphorylation of LHC II. Possible reasons for an involvement of tyrosine phosphorylation in light state transitions are proposed and discussed.

Balancing the two photosystems: photosynthetic electron transfer governs transcription of reaction centre genes in chloroplasts.

Chloroplasts are cytoplasmic organelles whose primary function is photosynthesis, but which also contain small, specialized and quasi-autonomous genetic systems. In photosynthesis, two energy converting photosystems are connected, electrochemically, in series. The connecting electron carriers are oxidized by photosystem I (PS I) and reduced by photosystem II (PS II). It has recently been shown that the oxidation reduction state of one connecting electron carrier, plastoquinone, controls transcription of chloroplast genes for reaction centre proteins of the two photosystems. The control counteracts the imbalance in electron transport that causes it: oxidized plastoquinone induces PS II and represses PS I; reduced plastoquinone induces PS I and represses PS II. This complementarity is observed both in vivo, using light favouring one or other photosystem, and in vitro, when site-specific electron transport inhibitors are added to transcriptionally and photosynthetically active chloroplasts. There is thus a transcriptional level of control that has a regulatory function similar to that of purely post-translational 'state transitions' in which the redistribution of absorbed excitation energy between photosystems is mediated by thylakoid membrane protein phosphorylation. The changes in rates of transcription that are induced by spectral changes in vivo can be detected even before the corresponding state transitions are complete, suggesting the operation of a branched pathway of redox signal transduction. These findings suggest a mechanism for adjustment of photosystem stoichiometry in which initial events involve a sensor of the redox state of plastoquinone, and may thus be the same as the initial events of state transitions. Redox control of chloroplast transcription is also consistent with the proposal that a direct regulatory coupling between electron transport and gene expression determines the function and composition of the chloroplast's extra-nuclear genetic system.

Photosynthetic electron flow regulates transcription of the psaB gene in pea (Pisum sativum L.) chloroplasts through the redox state of the plastoquinone pool.

Plants respond to changing light conditions by altering the stoichiometry between components of the photosynthetic electron transport chain of chloroplast thylakoids. We measured specific run-on transcription of the chloroplast genes psaB, psbA and rbcL in pea (Pisum sativum L.) seedlings grown under three different conditions of illumination: light selective for photosystem I (PSI-light); light selective for photosystem II (PSII-light); and a combination of PSI- and PSII-light (mixed light, ML). The transcriptional rate of the psaB gene increased under PSII-light and decreased under PSI-light, while the transcriptional rates of the psbA and rbcL genes were affected only in a non-specific way. Similar effects also occurred in plants grown under ML and switched to either PSI- or PSII-light for 4 h. Addition of the inhibitors of photosynthetic electron transport 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) influenced psaB transcription in isolated, illuminated chloroplasts: DCMU addition resulted in oxidation of the plastoquinone pool and decreased transcription of psaB; DBMIB addition resulted in reduction of the plastoquinone pool and increased transcription of psaB. The experimental results obtained in vivo and in vitro provide evidence for coupling between the redox state of plastoquinone and the rate of transcription of the psaB gene in pea.

Protein phosphorylation/dephosphorylation in the inner membrane of potato tuber mitochondria.

Inside-out inner mitochondrial membranes free of matrix proteins were isolated from purified potato tuber (Solanum tuberosum L.) mitochondria and incubated with ¿gamma-(32)PATP. Proteins were separated by SDS-PAGE and visualized by autoradiography. Phosphorylation of inner membrane proteins, including ATPase subunits, was strongly inhibited by the phosphoprotein phosphatase inhibitor NaF. We propose that an inner membrane phosphoprotein phosphatase is required for activation of the inner membrane protein kinase. When prelabelled inner membranes were incubated in the absence of ¿gamma-(32)PATP, there was no phosphoprotein dephosphorylation unless a soluble matrix fraction was added. This dephosphorylation was inhibited by NaF, but not by okadaic acid. We conclude that the mitochondrial matrix contains a phosphoprotein phosphatase that is responsible for dephosphorylation of inner membrane phosphoproteins.

Direct transcriptional control of the chloroplast genes psbA and psaAB adjusts photosynthesis to light energy distribution in plants.

Two photosystems, I and II, absorb and convert light energy in photosynthesis in chloroplasts of green plants. The genes psbA and psaAB of the cytoplasmic chloroplast genome encode core components of photosystem II and photosystem I, respectively. Here we show that the absolute amounts of photosystem I and photosystem II respond, in a complementary manner, to changes in light quality that preferentially excite each photosystem in mustard seedlings. We also show that the initial response to altered energy distribution is a change in the rates of transcription of psbA and psaAB. Changes in chlorophyll fluorescence emission in vivo suggest that the signal initiating this change is the oxidation-reduction state of plastoquinone, a component of the photosynthetic electron transport chain that connects photosystem I and photosystem II. The results are consistent with transcriptional effects observed previously with chloroplasts isolated in vitro and demonstrate that redox control of chloroplast transcription initiates long-term adjustments that compensate for imbalance in energy distribution and adapt the whole plant to altered light environments.

A mitochondrial model for premature ageing of somatically cloned mammals.

Cloned sheep have recently been discovered to have an unexpectedly advanced biological age. We propose that the explanation is a simple consequence of inheritance of acquired, free radical-induced cellular damage with somatic mitochondria that contribute to the mitochondrial population of cloned cells but not to zygotes produced by fertilization in normal sexual reproduction. Each increment of ageing in cloning experiments is therefore predicted to be maternally inherited. The hypothesis suggests practical ways of decreasing the effect. The hypothesis is itself a prediction of the recent proposal that mitochondria of the female germ line function primarily as genetic templates.

Truncated recombinant light harvesting complex II proteins are substrates for a protein kinase associated with photosystem II core complexes.

Previous studies directed towards understanding phosphorylation of the chlorophyll alb binding proteins comprising light harvesting complex II (LHC II) have concentrated on a single phosphorylation site located close to the N-terminus of the mature proteins. Here we show that a series of recombinant pea Lhcb1 proteins, each missing an N-terminal segment including this site, are nevertheless phosphorylated by a protein kinase associated with a photosystem II core complex preparation. An Lhch1 protein missing the first 58 amino acid residues is not, however, phosphorylated. The results demonstrate that the LHC II proteins are phosphorylated at one or more sites, the implications of which are discussed.

Protein synthesis by isolated pea mitochondria is dependent on the activity of respiratory complex II.

In isolated pea (Pisum sativum L.) mitochondria incorporation of 35S-methionine into newly synthesised proteins was influenced by the presence of site-specific inhibitors of the respiratory electron-transport chain. These effects were not produced by changes in the rate of respiratory electron transport itself nor by changes in ATP concentration. Protein synthesis was inhibited by inhibitors of ubiquinone reduction but not by inhibitors of ubiquinol oxidation. By the use of additional inhibitors at specific sites of the respiratory chain, different oxidation-reduction states were obtained for the different complexes in the electron-transport chain. It was found that electron transport through succinate:ubiquinone oxidoreductase (respiratory complex II) was specifically required for protein synthesis, even when all the other conditions for protein synthesis were satisfied. We suggest that a subunit of complex II, or a component closely associated with complex II, is involved in a regulatory system that couples electron transport to protein synthesis.

Two subunits of the F0F1-ATPase are phosphorylated in the inner mitochondrial membrane.

Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [gamma-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the delta'-subunit of the F1-ATPase and the b-subunit of the F0-ATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1 and F0 and regulate energy coupling in oxidative phosphorylation.

Phosphorylation controls the three-dimensional structure of plant light harvesting complex II.

The most abundant chlorophyll-binding complex in plants is the intrinsic membrane protein light-harvesting complex II (LHC II). LHC II acts as a light-harvesting antenna and has an important role in the distribution of absorbed energy between the two photosystems of photosynthesis. We used spectroscopic techniques to study a synthetic peptide with identical sequence to the LHC IIb N terminus found in pea, with and without the phosphorylated Thr at the 5th amino acid residue, and to study both forms of the native full-length protein. Our results show that the N terminus of LHC II changes structure upon phosphorylation and that the structural change resembles that of rabbit glycogen phosphorylase, one of the few phosphoproteins where both phosphorylated and non-phosphorylated structures have been solved. Our results indicate that phosphorylation of membrane proteins may regulate their function through structural protein-protein interactions in surface-exposed domains.