Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

The enormous metabolic plasticity of plants allows detoxificationof many harmful compounds that are generated during biosyntheticprocesses or are present as biotic or abiotic toxins in theirenvironment. Derivatives of toxic compounds such as glutathioneconjugates are moved into the central vacuole via ATP-bindingcassette (ABC)-type transporters of the multidrug resistance-associatedprotein (MRP) subfamily. The Arabidopsis genome contains 15AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) clustertogether in one of two major phylogenetic clades. We isolatedT-DNA knockout alleles in all four highly homologous AtMRP genesof this clade and subjected them to physiological analysis toassess the function of each AtMRP of this group. None of thesingle atmrp mutants displayed visible phenotypes under controlconditions. In spite of the fact that AtMRP1 and AtMRP2 hadbeen described as efficient ATP-dependent organic anion transportersin heterologous expression experiments, the contribution ofthree of the AtMRP genes (1, 11 and 12) to detoxification ismarginal. Only knockouts in AtMRP2 exhibited a reduced sensitivitytowards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides.AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophylldegradation since ethylene-treated rosettes of atmrp2 showedreduced senescence, and AtMRP2 expression is induced duringsenescence. This suggests that AtMRP2 is involved in vacuolartransport of chlorophyll catabolites. Vacuolar uptake studiesdemonstrated that transport of typical MRP substrates was reducedin atmrp2. We conclude that within clade I, only AtMRP2 contributessignificantly to overall organic anion pump activity in vivo.     

Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.     

Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members.

The nonstructural NS2 proteins of the prototype strain of minute virus of mice (MVMp) were previously shown to be involved in parvoviral DNA amplification as well as in efficient virus production in a host cell-specific manner (L. K. Naeger, N. Salomé, and D. J. Pintel, J. Virol. 67:1034-1043, 1993). NS2 polypeptides were also reported to participate in the cytotoxic activity of parvoviruses (C. Legrand, J. Rommelaere, and P. Caillet-Fauquet, Virology 195:149-155, 1993), for which transformed cells are preferential targets. To identify cellular partners of NS2 proteins, coimmunoprecipitation experiments were performed with various antibodies directed against the parvoviral products. Two cellular proteins with molecular masses of 30 and 32 kDa were found to associate in vivo with the NS2 polypeptides. From amino acid sequence homology, these NS2 partners were assigned to the 14-3-3 family of cellular proteins, showing at least partial identity with the epsilon and beta or zeta 14-3-3 isoforms. In agreement with this assignment, NS2-30/32-kDa protein immune complexes displayed an activating function for exoenzyme S in vitro, a hallmark of 14-3-3 polypeptides. Interactions with 14-3-3 proteins did not appear sufficient for NS2 functions, since they were not disrupted by NS2 C-terminal modifications that impaired virus replication. Binding of NS2 to 14-3-3 proteins was detected in various cells of mouse, rat, hamster, monkey, and human origin, irrespective of NS2 dispensability and host cell transformation or permissiveness. The ubiquitous 14-3-3 proteins were recently reported to associate with several other cellular or viral polypeptides involved in signal transduction and/or cell cycle regulation pathways (A. Aitken, Trends Biochem. Sci. 20:95-97, 1995). The NS2 products may connect with one of these pathways through their interaction with specific 14-3-3 polypeptides.     

Temperature and guanidine hydrochloride dependence of the structural stability of ribonuclease T1.

The thermal unfolding of ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.     

Micromorphological studies on seeds of orobanche species from the iberian peninsula and the balearic islands, and their systematic significance

BACKGROUND AND AIMS: Previous research has made clear the intrinsic taxonomic difficulties in identifying species in the genus Orobanche. The aim of this study, therefore, was to investigate the systematic utility of seed characteristics. METHODS: Light and scanning electron microscopy was used to examine the seeds of 33 taxa of Orobanche from the Iberian Peninsula and the Balearic Islands. KEY RESULTS: Characters such as size, shape and ornamentation of the seeds were not found to be very useful in differentiation of taxa; however, other characters of the epidermal seed coat cells proved to be very helpful in this respect. Ornamentation of the periclinal walls could be used to discriminate four morphological types. Other features related to the anticlinal walls of the cells, such as thickness, presence/absence of a narrow trough, or relative depth, all contributed to the characterization of a large number of species. CONCLUSIONS: The usefulness of micromorphological studies on seeds of Orobanche in relation to differentiating taxa is demonstrated, and a key is provided to distinguish species or groups of species.     

Biotic responses buffer warming‐induced soil organic carbon loss in Arctic tundra

Climate warming can result in both abiotic (e.g., permafrost thaw) and biotic (e.g., microbial functional genes) changes in Arctic tundra. Recent research has incorporated dynamic permafrost thaw in Earth system models (ESMs) and indicates that Arctic tundra could be a significant future carbon (C) source due to the enhanced decomposition of thawed deep soil C. However, warming‐induced biotic changes may influence biologically related parameters and the consequent projections in ESMs. How model parameters associated with biotic responses will change under warming and to what extent these changes affect projected C budgets have not been carefully examined. In this study, we synthesized six data sets over 5 years from a soil warming experiment at the Eight Mile Lake, Alaska, into the Terrestrial ECOsystem (TECO) model with a probabilistic inversion approach. The TECO model used multiple soil layers to track dynamics of thawed soil under different treatments. Our results show that warming increased light use efficiency of vegetation photosynthesis but decreased baseline (i.e., environment‐corrected) turnover rates of SOC in both the fast and slow pools in comparison with those under control. Moreover, the parameter changes generally amplified over time, suggesting processes of gradual physiological acclimation and functional gene shifts of both plants and microbes. The TECO model predicted that field warming from 2009 to 2013 resulted in cumulative C losses of 224 or 87 g/m², respectively, without or with changes in those parameters. Thus, warming‐induced parameter changes reduced predicted soil C loss by 61%. Our study suggests that it is critical to incorporate biotic changes in ESMs to improve the model performance in predicting C dynamics in permafrost regions.     

Synthesis of furanose glycals from furanose 1,2-diols and their cyclic thiocarbonate esters

Two new methods for the synthesis of furanoid glycals are described. Both procedures were shown to be faster and cheaper that those previously reported. Protected 1,2-dihydroxypento- and hexo-furanose derivatives with the -xylo, -gluco and -ribo, -allo configurations were used as starting material to afford the corresponding C-3,4 -threo and -erythro glycals derivatives.     

The role of tyrosine kinase in prostaglandin E2 and vanadate-evoked contractions in rabbit duodenum in vitro.

Prostaglandin E2 (PGE2) can interact with at least four cell surface receptors (EP1-EP4) in smooth muscle, which evokes a variety of intracellular responses depending on the G protein to which the cell surface receptors are coupled. The activation of G protein-coupled receptors and receptor tyrosine kinases can lead to the phosphorylation of tyrosine residues of various cellular proteins. The aim of this study was to examine the role of tyrosine phosphorylation in PGE2, vanadate and carbachol-evoked contractions. PGE2, vanadate, and carbachol induced contractile motor responses in the longitudinal smooth muscle of rabbit duodenum. PGE2-evoked contractions decreased in the presence of genistein or tyrphostin B44. PGE2-evoked contractions increased in the presence of vanadate. Vanadate-evoked contractions decreased in the presence of genistein. In contrast, tyrphostin 47 increased the vanadate-evoked contractions. Vanadate-evoked contractions were reduced in the presence of Ca2+-free solutions, verapamil, or indomethacin. U-73122 decreased PGE2-evoked contractions. Carbachol-evoked contractions decreased in the presence of genistein, tyrphostin B44 or tyrphostin 47. Our results suggest that PGE2, vanadate or carbachol-evoked contractions are mediated by protein tyrosine phosphorylation. Protein tyrosine phosphorylation might cause an increase in calcium influx through voltage-dependent channels and the release of prostaglandins in the longitudinal smooth muscle of the rabbit duodenum.     

Alpha-adrenergic influences on exocrine pancreatic secretion in the rabbit

Action of phenylephrine (35 micrograms/Kg/min) alone or previously blocked by phentolamine (100 micrograms/Kg/min) on exocrine pancreatic secretion of anaesthetized rabbits has been studied, in basal state or under stimulation by secretin (1 C.U./Kg/h) or by the octapeptide of cholecystokinin (OP-CCK) (0.15 Ivy dog units/Kg/h). Phenylephrine increased arterial pressure. This effect was blocked by phentolamine. However no variations were seen in pancreatic blood flow in any of the experimental conditions assayed. Phenylephrine produced a secretin-like effect on hydroelectrolytic secretion in basal conditions. This action was maintained after the infusion of secretin but not after OP-CCK. This effect was not blocked by phentolamine. Phenylephrine increased protein secretion in the basal state, an action that was blocked by phentolamine. After secretin or OP-CCK stimulation phenylephrine did not increase protein secretion. It is concluded that phentolamine blocks the effects of phenylephrine on acinar cells but not on ductular cells.