Mim1, a protein required for the assembly of the TOM complex of mitochondria

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.     

Niche construction through germination cueing: life-history responses to timing of germination in Arabidopsis thaliana

Germination responses to seasonal conditions determine the environment experienced by postgermination life stages, and this ability has potential consequences for the evolution of plant life histories. Using recombinant inbred lines of Arabidopsis thaliana, we tested whether life-history characters exhibited plasticity to germination timing, whether germination timing influenced the strength and mode of natural selection on life-history traits, and whether germination timing influenced the expression of genetic variation for life-history traits. Adult life-history traits exhibited strong plasticity to season of germination, and season of germination significantly altered the strength, mode, and even direction of selection on life-history traits under some conditions. None of the average plastic responses to season of germination or season of dispersal were adaptive, although some genotypes within our sample did exhibit adaptive responses. Thus, recombination between inbred lineages created some novel adaptive genotypes with improved responses to the seasonal timing of germination under some, but not all, conditions. Genetically based variation in germination time tended to augment genetic variances of adult life-history traits, but it did not increase the heritabilities because it also increased environmentally induced variance. Under some conditions, plasticity of life-history traits in response to genetically variable germination timing actually obscured genetic variation for those traits. Therefore, the evolution of germination responses can influence the evolution of life histories in a general manner by altering natural selection on life-history traits and the genetic variation of these traits.     

Vernalization sensitivity in Arabidopsis thaliana (Brassicaceae): the effects of latitude and FLC variation

Latitudinal variation in climate is predicted to select for latitudinal differentiation in sensitivity to the environmental cues that signal plants to flower at the appropriate time for a given climate. In Arabidopsis thaliana, flowering is promoted by exposure to cold temperatures (vernalization), and several vernalization pathway loci are known. To test whether natural variation in vernalization sensitivity could account for a previously observed latitudinal cline in flowering time in A. thaliana, we exposed 21 European accessions to 0, 10, 20, or 30 d of vernalization and observed leaf number at flowering under short days in a growth chamber. We observed a significant latitudinal cline in vernalization sensitivity: southern accessions were more sensitive to vernalization than northern accessions. In addition, accessions that were late flowering in the absence of vernalization were more sensitive to vernalization cues. Allelic variation at the flowering time regulatory gene FLC was not associated with mean vernalization sensitivity, but one allele class exhibited greater variance in vernalization sensitivity.     

Molecular identification and reconstitution of depolarization-induced exocytosis monitored by membrane capacitance

Regulated exocytosis of neurotransmitters at synapses is fast and tightly regulated. It is unclear which proteins constitute the "minimal molecular machinery" for this process. Here, we show that a novel technique of capacitance monitoring combined with heterologous protein expression can be used to reconstitute exocytosis that is fast (<0.5 s) and triggered directly by membrane depolarization in Xenopus oocytes. Testing synaptic proteins, voltage-gated Ca2+ channels, and using botulinum and tetanus neurotoxins established that the expression of a Ca2+ channel together with syntaxin 1A, SNAP-25, and synaptotagmin was sufficient and necessary for the reconstitution of depolarization-induced exocytosis. Similar to synaptic exocytosis, the reconstituted release was sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channel (Lc-, N-type), and depended nonlinearly on divalent cation concentration. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic exocytosis.     

Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter

Nucleotide-binding domains (NBD) are highly conserved constituents of ATP-binding cassette (ABC) transporters. Members of this family couple ATP hydrolysis to the transfer of various molecules across cell membranes. The NBD of the HlyB transporter, HlyB-NBD, was characterized with respect to its uncoupled ATPase activity, oligomeric state, and stability in solution. Experimental data showed that both the nature and pH of an assay buffer influenced the level of protein activity. Comparative analysis of protein stability and ATPase activity in various buffers suggests an inverse relationship between the two. The highest ATPase activity was detected in HEPES, pH 7.0. A kinetic analysis of the ATPase activity in this buffer revealed an enzyme concentration dependence and ATP-induced protein oligomerization. Assuming that the dimer is the active form of enzyme, at least half of the purified HlyB-NBD was estimated to be a dimer at 1.2 microM under the most optimal conditions for ATP hydrolysis. This is about 2 orders of magnitude lower than reported for other canonical ABC-ATPases. The maximum reaction velocity of 0.6 micromol/mg x min at 22 degrees C and the apparent kinetic constant K(app)(0.5) of 0.26 mM for ATP were determined for the dimerized HlyB-NBD. Gel filtration experiments with the wild-type protein and HlyB-NBD mutated in a key catalytic residue, H662A, provided further evidence for ATP-induced protein dimerization. ATPase activity experiments with protein mixtures composed of wild-type and the ATPase-deficient H662A mutant demonstrated that one intact NBD within a dimer is sufficient for ATP hydrolysis. This single site turnover might suggest a sequential mechanism of ATP hydrolysis in the intact HlyB transporter.     

Activation of camptothecin derivatives by conjugation to triple helix-forming oligonucleotides

Topoisomerase I (topo I) is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy. Camptothecins (CPTs) reversibly trap topo I in covalent complex with DNA but exhibit limited sequence preference. The utilization of conjugates such as triplex-forming oligonucleotides (TFOs) to target a medicinal agent (like CPT) to a specific genetic sequence and orientation within the DNA has been accomplished successfully. In this study, different attachment points of the TFO to CPT (including positions 7, 9, 10, and 12) were investigated and our findings confirmed and extended previous conclusions. Interestingly, the conjugates induced specific DNA cleavage by topo I at the triplex site even when poorly active or inactive CPT derivatives were used. This suggests that the positioning of the drug in the cleavage complex by the sequence-specific DNA ligand is able to stabilize the ternary complex, even when important interactions between topo I and CPT are disrupted. Finally, certain TFO-CPT conjugates were able to induce sequence-specific DNA cleavage with the topo I mutants R364H and N722S that are resistant to camptothecin. The TFO-CPT conjugates are thus valuable tools to study the interactions involved in the formation of the ternary complex and also to enlarge the family of compounds that poison topo I. The fact that an inactive CPT analogue can act as a topo I poison when appropriately coupled to a TFO provides a new perspective at the level of drug design.     

Crystal structure at 1.8 A resolution and identification of active site residues of Sulfolobus solfataricus peptidyl-tRNA hydrolase

The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 A resolution. Homologues of this enzyme are found in archaea and eucarya. Bacteria display a different type of peptidyl-tRNA hydrolase that is also encountered in eucarya. In solution, the S. solfataricus hydrolase behaves as a dimer. In agreement, the crystalline structure of this enzyme indicates the formation of a dimer. Each protomer is made of a mixed five-stranded beta-sheet surrounded by two groups of two alpha-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal alpha1 helices contributed by two protomers. Site-directed mutagenesis experiments were designed for probing the basis of specificity of the archaeal hydrolase. Among the strictly conserved residues within the archaeal/eucaryal peptidyl-tRNA hydrolase family, three residues, K18, D86, and T90, appear of utmost importance for activity. They are located in the N-part of alpha1 and in the beta3-beta4 loop. K18 and D86, which form a salt bridge, might play a role in the catalysis thanks to their acid and basic functions, whereas the OH group of T90 could act as a nucleophile. These observations clearly distinguish the active site of the archaeal/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine is believed to serve as the catalytic base.     

Effect of fenarimol on sterol biosynthesis in suspension cultures of bramble cells

Bramble suspension cultures normally contain Δ⁵-sterols (sitosterol, campesterol and isofucosterol). When the cells were grown in a medium supplemented with fenarimol, 14α-methyl sterols accumulated. Eight 14α-methyl sterols, including two new compounds, 4α,14α-dimethyl-stigmasta-8,Z-24(28)-dien-3β-ol and 14α-methyl-stigmasta-8,Z-24(28)-dien-3β-ol, were identified. Fenarimol probably inhibited the 14α-methyl demethylation. Cell lines growing permanently in 2 fenarimol-supplemented medium were obtained.     

Phenylphosphate synthase: a new phosphotransferase catalyzing the first step in anaerobic phenol metabolism in Thauera aromatica

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via para-carboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were investigated. Protein 1 (70 kDa) resembles the central part of classical phosphoenolpyruvate synthase which contains a conserved histidine residue. It catalyzes the exchange of free [(14)C]phenol and the phenol moiety of phenylphosphate but not the phosphorylation of phenol. Phosphorylation of phenol requires protein 1, MgATP, and another protein, protein 2 (40 kDa), which resembles the N-terminal part of phosphoenol pyruvate synthase. Proteins 1 and 2 catalyze the following reaction: phenol + MgATP + H(2)O-->phenylphosphate + MgAMP + orthophosphate. The phosphoryl group in phenylphosphate is derived from the beta-phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol (K(m), 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated severalfold by another protein, protein 3 (24 kDa), which contains two cystathionine-beta-synthase domains of unknown function but does not show significant overall similarity to known proteins. The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications.     

Thyroid cell proliferation in Graves' disease. Use of MIB-1 monoclonal antibody

OBJECTIVE: To measure thyroid cell proliferation in patients with Graves' disease (GD) before and during treatment with antithyroid drugs. STUDY DESIGN: Patients were assessed by fine needle aspiration biopsy before (n = 20) and after 4 (n = 19) and 12 months of treatment (n = 15) with propylthiouracil or methimazole. Cell proliferation index (CPI) was estimated by immunocytochemistry using MIB-1. CPI was studied in relation to the cytologic parameters of the smears; clinical parameters, such as Wayne's Clinical Index (WCI) and time without treatment; laboratory parameters, such as 131I uptake and dosage of serum free thyroxin and thyroid-stimulating hormone; and thyroid ultrasound. RESULTS: CPI varied from 0.00% to 25.00% before treatment, 0.00% to 23.00% at 4 months and 0.00% to 14.84% at 12 months. CPI median values were 6.50%, 4.30% and 3.30%, respectively (before and after 4 months and 12 months of treatment). CPI had a positive correlation with WCI and FT4 at 12 months of treatment. CONCLUSION: Thyroid CPI in GD varies from case to case. However, due to its decreasing pattern during follow-up and its positive correlation with thyrotoxicosis severity, CPI may indicate the functional status of the gland and contribute to a better understanding of GD.