Kinetic Evidence for the Uniport Mechanism Hypothesis in the Mitochondrial Tricarboxylate Transport System

The kinetics of the transport of citrate by the tricarboxylate transport system located in the inner mitochondrial membrane was studied in proteoliposomes containing the purified carrier protein, in order to verify the previously hypothesized mechanism of uniport (J. Bioenerg. Biomembr. 35, 133–140, 2003) and achieve some information on the kinetic properties of the carrier transport system. For this purpose, a mathematical model has been elaborated and the experimental data were analyzed according to it. The results indicate that the data actually fit with the uniport model, and hence it is confirmed that the carrier has a single binding site for its substrates and can oscillate between the inside and outside form, in both the free and substrate-bound states. The rearrangement of the free form is slower than the bound form in both directions. The dissociation constants for the internal substrate are at least one order of magnitude higher than the one for external citrate. As a consequence of these last two points, the rate of citrate transport by the carrier is much higher when it operates in exchange with another substrate than when it operates in net uniport.     

Mechanism of Folate Transport in Lactobacillus casei: Evidence for a Component Shared with the Thiamine and Biotin Transport Systems

Lactobacillus casei cells have been shown previously to utilize two separate binding proteins for the transport of folate and thiamine. Folate transport, however, was found to be strongly inhibited by thiamine in spite of the fact that the folate-binding protein has no measurable affinity for thiamine. This inhibition, which did not fluctuate with intracellular adenosine triphosphate levels, occurred only in cells containing functional transport systems for both vitamins and was noncompetitive with folate but competitive with respect to the level of folate-binding protein. Folate uptake in cells containing optimally induced transport systems for both vitamins was inhibited by thiamine (1 to 10 muM) to a maximum of 45%; the latter value increased to 77% in cells that contained a progressively diminished folate transport system and a normal thiamine system. Cells preloaded with thiamine could transport folate at a normal rate, indicating that the inhibition resulted from the entry of thiamine rather than from its presence in the cell. In a similar fashion, folate (1 to 10 muM) did not interfere with the binding of thiamine to its transport protein, but inhibited thiamine transport (to a maximum of 25%). Competition also extended to biotin, whose transport was strongly inhibited (58% and 73%, respectively) by the simultaneous uptake of either folate or thiamine; biotin, however, had only a minimal effect on either folate or thiamine transport. The nicotinate transport system was unaffected by co-transport with folate, thiamine, or biotin. These results are consistent with the hypothesis that the folate, thiamine, and biotin transport systems of L. casei each function via a specific binding protein, and that they require, in addition, a common component present in limiting amounts per cell. The latter may be a protein required for the coupling of energy to these transport processes.     

Evidence for a Shared Etiological Mechanism of Psychotic Symptoms and Obsessive-Compulsive Symptoms in Patients with Psychotic Disorders and Their Siblings

The prevalence of obsessive-compulsive disorder in subjects with psychotic disorder is much higher than in the general population. The higher than chance co-occurrence has also been demonstrated at the level of subclinical expression of both phenotypes. Both extended phenotypes have been shown to cluster in families. However, little is known about the origins of their elevated co-occurrence. In the present study, evidence for a shared etiological mechanism was investigated in 3 samples with decreasing levels of familial psychosis liability: 987 patients, 973 of their unaffected siblings and 566 healthy controls. The association between the obsessive-compulsive phenotype and the psychosis phenotype c.q. psychosis liability was investigated. First, the association was assessed between (subclinical) obsessive-compulsive symptoms and psychosis liability. Second, in a cross-sib cross-trait analysis, it was examined whether (subclinical) obsessive-compulsive symptoms in the patient were associated with (subclinical) psychotic symptoms in the related unaffected sibling. Evidence was found for both associations, which is compatible with a partially shared etiological pathway underlying obsessive-compulsive and psychotic disorder. This is the first study that used a cross-sib cross-trait design in patients and unaffected siblings, thus circumventing confounding by disease-related factors present in clinical samples.     

Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity

Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.     

A Requirement for FGF Signalling in the Formation of Primitive Streak-Like Intermediates from Primitive Ectoderm in Culture

Background

Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.

Methodology/Principal Findings

Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.

Conclusions/Significance

FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.     

Evidence for a Shared Nuclear Pore Complex Architecture That Is Conserved from the Last Common Eukaryotic Ancestor

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.Nearly all eukaryotic cells possess an extensive endomembrane system that is principally responsible for protein targeting and modification (1). The nucleus, the defining eukaryotic feature, is separated from the cytoplasm by a double bilayered nuclear envelope (NE)¹ that is contiguous with the rest of this endomembrane system via connections to the endoplasmic reticulum. Nuclear pore complexes (NPCs) fenestrate the NE, serving as the exclusive sites mediating exchange between the nucleoplasmic and cytoplasmic compartments. Macromolecules are chaperoned through the NPC by numerous transport factors. It has been proposed that the endomembrane system and nucleus have an autogenous origin (i.e. evolving from invaginations of an ancestral plasma membrane) and were established early in eukaryotic evolution (2).The composition of the NPC has been cataloged at ∼30 distinct nucleoporins (Nups) (3) for the yeast Saccharomyces cerevisiae (4) and vertebrates (5), two members of the Opisthokonta (animals, fungi, and closely related protists). Ultrastructural studies have identified objects morphologically similar (at a first approximation) to opisthokont NPCs in the other major eukaryote supergroups (6–8). However, very few data are available concerning the detailed NPC molecular composition and architecture for nearly all eukaryotic lineages, leaving a relatively narrow view of the “typical” NPC and its origins. A few examples of potential Nup orthologs beyond the opisthokonts have been reported, leading to the suggestion that substantial portions of the NPC may have an ancient, pre-last common eukaryotic ancestor (LCEA) origin (9). However, a more extensive study has concluded that LCEA possessed a primitive ancestral NPC that passed few components to its modern descendants (10).In yeast and vertebrates, the NPC consists of an eight-spoked core surrounding a central tube that serves as the conduit for macromolecular exchange. Each spoke can be divided into two similar nucleoplasmic and cytoplasmic halves. The eight spokes connect to form several coaxial rings: the membrane rings, the two outer rings at the nucleoplasmic and cytoplasmic periphery, and the two adjacent inner rings (11). Groups of Nups that we term “linker Nups” are attached between both sets of outer and inner rings. Another group of related proteins, collectively termed phenylalanine-glycine (FG) Nups, are largely exposed on the inner surface of the spokes and anchored either to the inner rings or to the linker Nups (11).Opisthokont Nups can be grouped into three structural classes (11, 12). The first class comprises membrane-bound proteins that anchor the NPC into the NE. The second class is the core scaffold Nups; these proteins constitute the bulk of the NPC mass, form the central tube, and provide the scaffold for the deployment of the third class of Nups across both faces of the NPC. The core scaffold Nups are remarkably restricted at the structural level and contain only three distinct arrangements of 2-fold types: proteins dominated by an α-solenoid fold (also termed a helix-turn-helix repeat domain), proteins consisting of a β-propeller fold, and finally proteins composed of an amino-terminal β-propeller fold followed by a carboxyl-terminal α-solenoid fold (which we here term a β-α structure) (12). FG Nups comprise the third class. These Nups carry multiply repeated degenerate “Phe-Gly” motifs (FG repeats) separated by hydrophilic or charged residues that form large unstructured domains. Each FG Nup also contains a small structured domain (often a coiled coil motif) that serves as the anchor site for interaction with the remainder of the NPC.Many transport factors belong to a structurally related protein family collectively termed karyopherins (Kaps) (13, 14). Transport across the NPC depends on the interactions between Kaps, cargo molecules, and the disordered repeat domains of FG Nups; the latter are thought to form the selective barrier for nucleocytoplasmic transport, guiding the Kap·cargo complexes (and other transport factors) through the central tube while excluding other macromolecules (for reviews, see Refs. 3 and 15–22).Significantly we have previously noted that the fold composition and arrangement of many of the core scaffold Nups are shared with proteins that form coating structures that participate in the generation and transport of vesicles between different endomembrane compartments; significantly many vesicle coating complex proteins and NPC scaffold Nups share an α-solenoid fold, β-propeller fold, or β-α structure (12, 23–28). These similarities gave rise to the “protocoatomer hypothesis,” which suggests a common ancestry for the NPC and these vesicle coat complexes. However, it is unclear how many, if any, of these particular core scaffold Nups are widely conserved, and hence it is unclear how general this potential relationship is throughout the Eukaryota. Thus, two scenarios are possible. The first is that the coatomer-like proteins are only found in a subset of the eukaryotes (including the opisthokonts), indicating that they are a relatively recent acquisition of only some eukaryotes and are not a general feature of all NPCs. The second is that the coatomer-like proteins are conserved in all eukaryotes, providing strong support to the protocoatomer hypothesis. To directly address this issue we characterized the NPC of Trypanosoma brucei, a highly divergent but experimentally tractable organism, using proteomics. The resulting data indicate an ancient origin for the majority of the NPC components and shed light on the origin of LCEA itself.     

Evidence for Shared Cognitive Processing of Pitch in Music and Language

Language and music epitomize the complex representational and computational capacities of the human mind. Strikingly similar in their structural and expressive features, a longstanding question is whether the perceptual and cognitive mechanisms underlying these abilities are shared or distinct – either from each other or from other mental processes. One prominent feature shared between language and music is signal encoding using pitch, conveying pragmatics and semantics in language and melody in music. We investigated how pitch processing is shared between language and music by measuring consistency in individual differences in pitch perception across language, music, and three control conditions intended to assess basic sensory and domain-general cognitive processes. Individuals’ pitch perception abilities in language and music were most strongly related, even after accounting for performance in all control conditions. These results provide behavioral evidence, based on patterns of individual differences, that is consistent with the hypothesis that cognitive mechanisms for pitch processing may be shared between language and music.     

Equilibrium and Kinetic Studies of Phosphate Removal from Solution onto a Hydrothermally Modified Oyster Shell Material

Phosphate removal to a hydrothermally modified fumed silica and pulverized oyster shell material for use in wastewater treatments were made. Sorption data modeling (pH’s 3–11, P concentrations of 3, 5, 10, 15, 20, & 25 mg/L, and at an ambient temperature of 23°C) indicate that an optimal removal of P occurs at pH 11. Three kinetic models were also applied (a pseudo-first-order Lagergren kinetic model, a pseudo-second-order (PSO) kinetic and Elovich) and indicate that a PSO model best describes P-removal. In addition, an application of the Weber and Morris intra-particle diffusion model indicates that external mass transfer and intra-particle diffusion were both involved in the rate-determining step. Langmuir, Freundlich modeling of the sorption data also indicate that the heterogeneous Freundlich sorption site model best describes the data although Langmuir data also fit with data tailing suggesting data are not linear. The data collected indicates that the hydrothermally modified fumed silica and pulverized oyster shell material is suitable for use in wastewater treatment, with P-removal to the solids being preferential and spontaneous.     

Crystal Structure,SAXS and Kinetic Mechanism of Hyperthermophilic ADP-Dependent Glucokinase from Thermococcus litoralis Reveal a Conserved Mechanism for Catalysis

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: II. Evidence for the Participation of Uridine Diphosphoxylose and Free Xylose as Intermediates

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis.     

Equilibrium binding of thioredoxin fB to chloroplastic fructose bisphosphatase. Evidence for a thioredoxin site distinct from the active site

Thioredoxin fB, the protein activator of chloroplastic fructose 1,6-bisphosphatase, strongly binds its target enzyme with a stoichiometry of one protein dimer per enzyme tetramer. The thioredoxin binding site is distinct from the active site and the dissociation constant of the protein-enzyme complex has the extremely small value of 769 nM at pH 7.5. This interaction involves both ionic and hydrophobic contributions and is enhanced by a pH increase from 7 to 8. These results suggest that the above molecular properties may be involved in the light activation of chloroplastic fructose bisphosphatase.     

System of Kinetic Equations for Collisionless Space Plasma in the Approximation of Field-Aligned Force Equilibrium for Electrons

A system of kinetic equations describing relatively slow large-scale processes in collisionless magnetoplasma structures with a spatial resolution on the order of the proton thermal gyroradius is derived. The system correctly takes into account the electrostatic effects in the approximation of field-aligned force equilibrium for electrons. The plasma is considered quasineutral, and the magnetic field is described by the Ampère equation. The longitudinal component of the electric field is found explicitly from the equality of the field-aligned component of the electric force acting on plasma electrons and the divergence of the electron pressure tensor. The electric field component orthogonal to the magnetic field is determined by the distributions of the number densities, current densities, and stress tensors of all plasma species in the instantaneous long-range approximation described by a system of time-independent elliptic equations. Versions of the system of equations adapted to the case of magnetized electrons described by the Vlasov equation in the drift approximation, as well as to the case in which all plasma species are magnetized, are derived. The resulting systems of equations allow creating numerical models capable of describing large-scale processes in nonuniform collisionless space plasma.     

Evidence for a Conjugation-Like Mechanism of DNA Transfer in Helicobacter pylori

Many strains of Helicobacter pylori are naturally competent for transformation in vitro. Since there is a high degree of genetic variation among H. pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms. Studies were done with H. pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase. In each experiment, such matings produced progeny with the markers of both parents. Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional. DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so. For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional. DNase-treated cell-free supernatants also did not transform, ruling out transduction. These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H. pylori cells.     

Mixing Nulliparous and Multiparous Women in Randomised Controlled Trials of Preeclampsia Prevention Is Debatable: Evidence from a Systematic Review

Background

Nulliparity is a major risk factor of preeclampsia investigated in numerous trials of its prevention.

Objective

We aimed to assess whether these trials considered nulliparity in subject selection or analysis of results.

Search Strategy

01 April 2013 search of MEDLINE via PubMed, EMBASE and the Cochrane Library. 01 April 2013 search of trials registered in Clinicaltrials.gov.

Selection Criteria

Randomised controlled trials and metaanalyses of preeclampsia prevention with no restriction to period of publication or language. Metaanalyses were selected to fully identify relevant trials.

Data Collection and Analysis

One reader appraised each selected article/registered protocol using a pretested, standardized data abstraction form developed in a pilot test. For each article, he recorded whether both nulliparous and multiparous were included and, in case of mixed populations, whether randomisation was stratified, and whether subgroup analyses had been reported. For registered protocols, he only assessed whether it was planned to include mixed populations.

Main Results

88 randomised controlled trials were identified, representing 83,396 included women. In 58 of the 88 articles identified (65.9%), preeclampsia was the primary outcome. In 31 of these (53.4%), the investigation combined nulliparous and multiparous women; only two reports in 31 (6.5%) stated that randomisation was stratified on parity and only four (12.9%) described a subgroup analysis by parity. Of the 30 registered trials, 20 (66.6%) planned to include both nulliparous and multiparous women.

Conclusion

Parity is largely ignored in randomised controlled trials of preeclampsia prevention, which raises difficulties in interpreting the results.     

Equilibrium and Kinetic Studies on the Removal of Basic Violet 10 from Aqueous Solutions Using Activated Carbons Prepared from Industrial Wastes

Low-cost adsorbents prepared from industrial wastes such as sugarcane bagasse (BC), rice husk (RC), and textile waste cloth (TC) are identified as suitable sorbents for removing basic violet 10 (BV). Fourier transform infrared (FTIR) and scanning electron microscopic (SEM) studies were carried out to characterize the prepared sorbents. The effects of dosage, time, and pH on dye removal were examined. It was observed that BV sorption takes place in acidic, neutral, and alkaline aqueous solutions. The work discussed the best-fit sorption isotherms among Freundlich and Langmuir, in addition to the reaction- and diffusion-based kinetic models. Based on the data obtained, it was found that the BV sorption took place in acidic, neutral, and alkaline aqueous solutions and sorption kinetics found to be controlled by pseudo-second-order and pore diffusion models. Also, thermodynamic parameters such as ΔG ^o, ΔH ^o, ΔS ^o, and E _a were calculated in order to understand the nature of the sorption process. The maximum dye removal capacity (DRC) was found to be 5608, 1244, and 27,495 mg/kg for BC, RC, and TC, respectively. Collectively, it can be concluded that the activated carbon sorbents, prepared from the named wastes, can used to adequately remove the basic violet dye from its aqueous waste solution.     

How Covalent Heme to Protein Bonds Influence the Formation and Reactivity of Redox Intermediates of a Bacterial Peroxidase

The most striking feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO) is the existence of covalent bonds between the prosthetic group and the protein, which has a strong impact on their (electronic) structure and biophysical and chemical properties. Recently, a novel bacterial heme peroxidase with high structural and functional similarities to LPO was described. Being released from Escherichia coli, it contains mainly heme b, which can be autocatalytically modified and covalently bound to the protein by incubation with hydrogen peroxide. In the present study, we investigated the reactivity of these two forms in their ferric, compound I and compound II state in a multi-mixing stopped-flow study. Upon heme modification, the reactions between the ferric proteins with cyanide or H₂O₂ were accelerated. Moreover, apparent bimolecular rate constants of the reaction of compound I with iodide, thiocyanate, bromide, and tyrosine increased significantly and became similar to LPO. Kinetic data are discussed and compared with known structure-function relationships of the mammalian peroxidases LPO and myeloperoxidase.     

Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.     

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.