Maintenance of meristem activity under stress: is there an interplay of RSS1-like proteins with the RBR pathway?

Plants have acquired rapid responses to a constantly changing environment. These adaptive and protective responses are the result of a complex signalling network regulating different aspects, ranging from ion homeostasis to cell cycle control. It is well established that stress inhibits cell division, which negatively impacts plant growth and development and hence results in biomass decrease and yield loss. Therefore understanding the link between stress perception and cell cycle control would allow development of new crops with increased productivity when subjected to stress. However, studies on cell cycle control under stress have been limited to well-known regulators of the cell cycle such as cyclins and stress-related phytohormone integrators. The recent discovery of RSS1, a novel intrinsically unstructured protein of rice, opened up new insights into how stress perception can be connected with cell cycle control in meristematic zones. Whereas RSS1 is well conserved among other plant lineages, eudicots present proteins sharing little sequence homology with RSS1. Here, we discuss how RSS1-like proteins might also be functional in dicots, and possibly act through the retinoblastoma-related pathway to regulate both S-phase transition and cell fate in meristems.

Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis.

Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of -14 degrees C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at -22.5 degrees C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (-18.5 degrees C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.

Solution studies of elongation factor Tu from the extreme halophile Halobacterium marismortui.

The activity, stability and structure in solution of polypeptide elongation factor hEF-Tu from Halobacterium marismortui have been investigated. The protein is stable in aqueous solutions only at high concentrations of NaCl, KCl or ammonium sulphate, whereas it is more active in exchanging GDP at lower salt concentrations. It is more active and stable at lower pH values than is non-halophilic EF-Tu. The structure in solution of the protein was determined by complementary density, ultracentrifugation, dynamic light-scattering and neutron-scattering measurements. The protein has large hydration interactions, similar to those of other halophilic proteins: 0.4 (+/- 0.1) g of water and 0.20 (+/- 0.05) g of KCl associated with 1 g of protein, with a water/KCl mass ratio always remaining close to 2. The kinetics of inactivation at low salt concentrations showed a stabilizing effect of NaCl when compared to KCl. At low salt concentration, inactivation, protein unfolding and aggregation were strongly correlated. The results suggest that the stabilization model proposed for halophilic malate dehydrogenase by Zaccai et al., involving extensive protein interactions with hydrated salt ions, is also valid for hEF-Tu.

Functional determinants of the Epstein-Barr virus protease.

Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.

Induction of H(2)O(2) synthesis by beta-glucan elicitors in soybean is independent of cytosolic calcium transients.

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.

Signal transduction via both human low-affinity IgG Fc receptors, Fc gamma RIIa and Fc gamma RIIIb, depends on the activity of different families of intracellular kinases.

In contrast to IgG Fc receptor II (Fc gamma RIIa), the function of Src-family kinases, Syk and phosphoinositide 3-kinase (PI3K) in signal transduction of glycosylphosphatidylinositol-anchored Fc gamma RIIIb has not been analyzed in detail. Therefore pharmacological inhibitors were used to define the role of specific kinases in signalling of Fc gamma RIIa and Fc gamma RIIIb in myeloid cells. We demonstrate that the broad tyrosine kinase inhibitor genistein, the Src-family kinase inhibitor PP2, and the Syk kinase inhibitor piceatannol inhibit [Ca2+]i rise induced by both low-affinity Fc gamma R in neutrophils. Genistein and PP2 additionally prevent Fc gamma R-triggered hydrogen peroxide generation. In contrast, wortmannin, a PI3K inhibitor, which blocks Fc gamma RIIIb activation completely, abolishes Fc gamma RIIa-mediated [Ca2+]i flux only in the beginning. In addition, herbimycin A, a further specific inhibitor of Src-family kinases leads to a delayed Fc gamma RIIa-induced [Ca2+]i rise in THP-1 cells. In summary, our data demonstrate differences between both low-affinity IgG Fc receptors, and provide evidence for an essential role of Src-family kinases, Syk and PI3K in Fc gamma RIIIb-mediated signalling.

[3H]diazepam binding sites on chick neurons in primary culture.

High affinity [3H]diazepam binding sites were identified on neurons prepared from the hemispheres of 8-day-old chick embryos and grown in serum-containing or serum-free medium. Clonazepam (IC50 = 3 nM) was more potent than Ro 5-4864 (IC50 greater than 1000 nM) in displacing [3H]diazepam binding. GABA and pentobarbital, in the presence of chloride ions were able to stimulate [3H]diazepam binding synergistically. These interactions were found to be comparable to those observed in mammalian brain.

Solution structure of glyceraldehyde-3-phosphate dehydrogenase from Haloarcula vallismortis.

The subunit molecular mass of glyceraldehyde-3-phosphate dehydrogenase from the extreme halophile Haloarcula vallismortis (hGAPDH) was determined by mass spectrometry to be 35990 +/- 80 daltons, similar to other GAPDHs. Complementary density, sedimentation and light scattering experiments showed the protein to be a tetramer that binds 0.18 +/- 0.10 gram of water and 0.07 +/- 0.02 gram of KCl per gram of protein, in multimolar KCl solutions. At low salt (below 1 M), the tetramer dissociated into unfolded monomers. This is the third halophilic protein for which solvent interactions were measured. The extent of these interactions depends on the protein, but all form an invariant particle, in multimolar NaCl or KCl solutions, that binds a high proportion of salt when compared to non-halophilic proteins.

The psychosocial impact of human papillomavirus infection: implications for health care providers.

The American Social Health Association (ASHA) surveyed people with human papillomavirus (HPV) about their experiences with the disease and its effect on their lives. A sample of 837 was chosen from the subscribers to HPV News, ASHA's quarterly journal for people with HPV. Of the sample, 489 returned completed surveys, which addressed medical history, health care experiences, personal impact, and demographic information. Data analysis was descriptive. Data illustrated that the psychosocial impact of HPV can be serious. More than three-quarters of respondents reported feelings of depression and anger, and two-thirds feelings of shame. Sexual enjoyment and activity were also negatively affected by HPV. Additionally, respondents expressed dissatisfaction with the diagnosing health care providers' counselling on emotional and sexual issues. These results may be instructive to those delivering health services by providing insight into the significant personal impact of HPV on those infected.

Testing an electronic documentation system.

Testing of newly automated clinical documentation system functions in patient care areas is integral to successful automation. Trialing the new functions in the patient care environment can be accomplished by use of alpha and beta patient care areas. Throughout the trialing of functions, involvement and feedback from patient care area managers and staff is crucial for successful automation.

[Assessment of reasons for overactive bladder treatment change]. / Evaluación de los motivos del cambio de tratamiento para la vejiga hiperactiva.

OBJECTIVES: although efficacious, some patients do not respond optimally to overactive bladder (OAB) treatment. The objective of this study was to identify the reasons why some patients do not respond and to look for reasons for changes in treatment and patient satisfaction with the new treatment. MATERIALS AND METHODS: epidemiological, cross-sectional, non-interventional study to determine the reasons for OAB treatment switching and satisfaction with such OAB treatment switch. OAB patients (OAB-V8≥8), 18 years or more, who had modified their treatment during the previous 3-4 months, were recruited. Demographic data, symptoms, previous, current and concomitant treatments, reasons for treatment switch, clinical global impression (CGI) on disease severity and symptom improvement, Morinsky Green questionnaire, satisfaction with treatment, treatment preference and treatment benefit scale (TBS) were compared. RESULTS: out of 3,365 successive patients, 2,038 (61%) were eligible (61.1±11.2 years; 77% women). The physician decided to switch in 69% of the cases and 31% of patients asked for a change in treatment. Reasons for switching were lack of clinical benefit (60%), side effects (24%), patients' request (8%), non-compliance (6%) and other (2%). 52% of patients complied with new treatment. According to the CGI, 65.4% showed improvement with respect to their previous treatment. 60% were quite/very satisfied with current treatment, 91% preferred it to their previous treatment and 93% reported that their symptoms had improved. CONCLUSIONS: the lack of clinical benefit is the main reason for changing OAB treatment. Most of the patients that switched prefer their new treatment.

Amphipols from A to Z.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

Halophilic adaptation of enzymes.

It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics. In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed. We then describe how thermodynamic observations, such as parameters pertaining to solvent-protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins. The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly.

Probing protein-sugar interactions.

We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents.

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Characterization of trans-splicing in Euglenoids.

We have looked for trans-splicing of nuclear mRNAs in several Euglenoid species. In Cyclidiopsis acus, Phacus curvicauda, Rhabdomonas costata and Menoidium pellucidum we showed that several premRNAs chosen at random are matured by a transsplicing process: we identified SL-RNA genes whose 5' ends (SLs for spliced leader-sequences) were transferred to the 5' extremities of mRNAs. The SL-RNA genes are located on repeated DNA fragments which also encode 5S rRNA in P. curvicauda and C. acus. The potential secondary structures of SL-RNAs are compared to those previously characterized in two other Euglenoids: Euglena gracilis and Entosiphon sulcatum. In another Euglenoid species, Distigma proteus, since none of the mRNAs examined were trans-spliced, it is possible that trans-splicing does not occur. Phylogeny based on 5S rRNA sequences suggests that the species which have, or have had, chloroplasts (E. gracilis, P. curvicauda, C. acus) diverged early from the others.

A central role for water in the control of the spin state of cytochrome P-450scc.

A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73] demonstrated two conformations (A and B) of cytochrome P-450scc (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The conformation found at the lowest temperatures (form A) displays a six-ligand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P., Lange, R., Krüger, V. & Stier, A. (1994) Biochemistry 33, 12920-12929]. Analytical centrifugation shows that the oligomeric composition of SCC is the same for the A and the B conformers. However, as revealed by fourth-derivative ultraviolet spectroscopy, the two conformers differ in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conformations was investigated using the pressure-jump technique under various pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxation kinetics corresponding to the high-spin to low-spin transition. Analysis of the activation volumes suggested a dissociative mechanism for the A conformer (+45 ml/mol), and an associative mechanism for the B conformer (-39 ml/mol). Applying osmotic stress to the A form changed its kinetic characteristics to those of the B form. These results are consistent with a model comprising a solvent intake (ten water molecules) between the B and the A conformers and protonation of their respective high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining structure.

Reversible dissociation and unfolding of dimeric creatine kinase isoenzyme MM in guanidine hydrochloride and urea.

The unfolding of dimeric cytoplasmic creatine kinase (MM) by guanidine hydrochloride and by urea has been investigated using activity measurements, far-ultraviolet circular dichroism, sedimentation velocity and fluorescence energy transfer experiments to monitor global structural changes. Intrinsic (cysteine and tryptophan residues) and extrinsic probes (1-anilinonaphthalene-8-sulfonate) were also used. The reversibility of the unfolding was checked by monitoring activity and tryptophan fluorescence. The unfolding of creatine kinase in guanidine hydrochloride is a reversible multistep process, as suggested by the non-coincidence of denaturation curves at equilibrium. Inactivation of the dimer precedes its dissociation into two monomers and an intermediate state was identified during the unfolding of the monomer. This intermediate state is characterized by a relatively high degree of secondary structure (as shown by far-ultraviolet circular dichroism), of compactness (as shown by fluorescence energy transfer measurements and sedimentation experiments), a fluctuating tertiary structure (as shown by near-ultraviolet circular dichroism) and a strong affinity for anilinonaphthalene sulfonate (as demonstrated by fluorescence). These results clearly indicate that the intermediate state detected possesses some of the properties of a molten globule. In urea, the unfolding pathway is reversible but differs from that observed in guanidine hydrochloride. Indeed inactivation, dissociation and loss of tertiary structure are coincident but the ellipticity curve is slightly shifted to a higher urea concentration. The dimer is dissociated into two expanded monomers possessing some secondary structure which is progressively lost at a higher urea concentration (6.4M). These results show that guanidine hydrochloride is approximately six times more effective than urea for inactivation and dissociation, underlining the fact that electrostatic interactions are very important in the stabilization of the active site and of the dimeric state.

Aggregation of VSV M protein is reversible and mediated by nucleation sites: implications for viral assembly.

Purified M protein of VSV has been reported to aggregate at low NaCl concentration. Using light scattering, analytical centrifugation, and electron microscopy (EM), we have studied this phenomenon. Our results demonstrate that self aggregation of M protein can be reversed by increasing the salt concentration. Below 250 mM NaCl, there is an equilibrium between aggregates and monomeric M protein. Most importantly, we demonstrate that aggregation only occurs in the presence of nucleation sites and that these sites are sensitive to trypsin. We have found conditions under which these nucleation sites can be eliminated, after which M remains soluble even at low salt concentration. Finally, using EM, we show that the aggregates of purified M protein share common structural aspects with the previously described internal "cigar" around which the nucleocapsid is wrapped. These new results help to explain why M is a soluble protein in the cytoplasm of the infected cell just up to the moment that it is integrated into the budding virion.