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.

Biophysical characterization of the C-propeptide trimer from human procollagen III reveals a tri-lobed structure.

Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

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.

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro.

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

On the domain structure and the polymerization state of the sendai virus P protein.

The phosphoproteins (P) of paramyxoviruses and rhabdoviruses are cofactors of the viral polymerase (L) and chaperones of soluble nucleoprotein preventing its polymerization and nonspecific binding to cellular RNA. The primary sequences of six paramyxovirus P proteins were compared, and although there was virtually no sequence similarity, there were two regions with similar secondary structure predictions in the C-terminal part of P: the predicted multimerization domain and the X-protein, the sequence that binds to N in the N:RNA template. The C-terminal part of the Sendai virus P protein, the multimerization domain including the binding site for the polymerase, and the X-protein were expressed in Escherichia coli. All three polypeptides folded with secondary structures similar to those predicted. The C-terminal part of P is a very elongated molecule with most of its length encompassing the multimerization domain. Both the multimerization domain and the C-terminal part of P were found to form tetramers, whereas the X-protein was monomeric.

Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70.

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.

Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species.

The kinetics of refolding of guanidinium chloride-denatured rabbit MM-creatine kinase was investigated. Recovery of enzymatic activity is biphasic, depending on the temperature but not on the protein or DTT concentration. Only 45% of the original, active dimeric form is recovered even after several hours of refolding. The reactivation yield is limited by the accumulation of a highly stable but nonproductive monomeric species. The ratio of "correct" to "incorrect" forms depends on the duration of exposure to the denaturant, which may be consistent with the existence of a heterogeneous population of unfolded states with regard to proline isomerization. The first fast reaction observed during renaturation results in the appearance of collapsed monomeric states, displaying features of a pre-molten globule state. These burst species are rapidly transformed into more structured monomers resembling a molten globule state possessing a partially folded C-terminal domain. A proportion of these latter transient intermediates (45%) associates into an active dimer, while the remainder (55%) is trapped by reshuffling in a monomeric dead-end product. Our results strongly indicate that (i) the dimeric state is a prerequisite for the expression of catalytic activity, (ii) the kinetic intermediates of refolding are very similar to those observed during equilibrium unfolding, and (iii) refolding of creatine kinase in these conditions is limited by the accumulation of inactive misfolded nondimerizable monomer.

Effect of thoracic epidural anaesthesia on ventilation-perfusion distribution and intrathoracic blood volume before and after induction of general anaesthesia.

BACKGROUND: Gas exchange is impaired during general anaesthesia due to development of shunt and ventilation-perfusion mismatching. Thoracic epidural anaesthesia (TEA) may affect the mechanics of the respiratory system, intrathoracic blood volume and possibly ventilation-perfusion (VA/Q) distribution during general anaesthesia. METHODS: VA/Q relationships were analyzed in 24 patients undergoing major abdominal surgery. Intrapulmonary shunt (Qs/QT), perfusion of "low" VA/Q areas, ventilation of "high" VA/Q regions, dead space ventilation and mean distribution of ventilation and perfusion were calculated from the retention/excretion data of six inert gases. Intrathoracic blood volume (ITBV) and pulmonary blood volume (PBV) were determined with a double indicator technique. Recordings were made before and after administration of 8.5 +/- 1.5 ml bupivacaine 0.5% (n = 12) or 8.3 +/- 1.8 ml placebo (n = 12) into a thoracic epidural catheter and after induction of general anaesthesia. RESULTS: Before TEA, Qs/QT was normal in the bupivacaine group (2 +/- 2%) and the placebo group (2 +/- 3%). TEA covering the dermatomal segments T 12 to T 4 had no effect on VA/Q relationships, ITBV and PBV. After induction of general anaesthesia Qs/QT increased to 8 +/- 4% (bupivacaine group, P < 0.05 and to 7 +/- 2% (placebo group, P < 0.05). ITBV and PBV decreased significantly to the same extent in the bupivacaine group and the placebo group. CONCLUSIONS: TEA has no effect on VA/Q distribution, gas exchange and intrathoracic blood volume in the awake state and does not influence development of Qs/QT and VA/Q inequality after induction of general anaesthesia.

An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

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.

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.

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.