Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.     

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.     

Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells.

The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells. The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker. Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex. VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins. Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence. Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced. We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking.     

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.     

Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis

Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

PDB2MultiGIF: A Web Tool to Create Animated Images of Molecules

A new web tool, PDB2MultiGIF (http://www.dkfz-heidelberg.de/spec/pdb2mgif/),which converts the topological information (atom types, 3D coordinates, molecular connectivity) of molecules (given in PDB format [1]) to a series of animated images (in GIF Format) [2] is described. The molecular visualisation program RASMOL [3] is used to generate the images.Electronic Supplementary Material available.     

Phylogenetic analysis of freshwater sponges provide evidence for endemism and radiation in ancient lakes

Morphologic and phylogenetic analysis of freshwater sponges endemic to lakes in Central Sulawesi, Siberia and South-East Europe is presented. We also analyzed several cosmopolitan sponge species from Eurasia and North America and included sponge sequences from public databases. In agreement with previous reports [Addis, J.S., Peterson, K.J., 2005. Phylogenetic relationships of freshwater sponges (Porifera, Spongillina) inferred from analyses of 18S rDNA, COI mtDNA, and ITS2 rDNA sequences. Zool. Scr. 34, 549-557], the metaniid sponge Corvomeyenia sp. was the most deeply branching species within a monophyletic lineage of the suborder Spongillina. Pachydictyum globosum (Malawispongiidae) and Nudospongilla vasta (Spongillidae), two morphologically quite distinct species from Sulawesi were found in a joint clade with Trochospongilla (Spongillidae) rendering Trochospongilla paraphyletic. Furthermore, Ochridaspongia sp., another Malawispongiidae, clustered far away from that clade, together with Ephydatia fluviatilis, making the latter family polyphyletic. The Lubomirskiidae endemic to Lake Baikal, Lubomirskia abietina, Baikalospongia bacillifera, B. intermedia, and Swartschewskia papyracea formed a well-supported clade that was most closely linked to the genus Ephydatia (99.9% identity over a total length of 2169 concatenated nucleotide positions). Our study indicates the frequent and independent origin of sponge species endemic to different freshwater ecosystems from a few cosmopolitan founder species. The highly specific primer sets newly developed here facilitate work on the molecular phylogeny and DNA barcoding of sponges.     

Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth

In many apicomplexan parasites the entry of electrons from NADH into the electron transport chain is governed by type II NADH dehydrogenases (NDH2s) instead of a canonical complex I. Toxoplasma gondii expresses two NDH2 isoforms, TgNDH2-I and TgNDH2-II with no indication for stage-specific regulation. We dissected the orientation of both isoforms by using a split GFP assay and a protease protection assay after selective membrane permeabilization. The two approaches revealed that both TgNDH2 isoforms are internal enzymes facing with their active sites to the mitochondrial matrix. Single knockout mutants displayed a decreased replication rate and a reduced mitochondrial membrane potential, which were both more severe in the Tgndh2-II-deleted than in the Tgndh2-I-deleted mutant. Complementation with a myc-tagged, ectopic copy of the deleted gene restored the growth rate and the mitochondrial membrane potential. However, an overexpression of the remaining intact isoform could not restore the phenotype, suggesting that the two TgNDH2 isoforms are non-redundant and possess functional differences. Together, our studies indicate that although TgNDH2-I and TgNDH2-II are individually non-essential, the expression of both internal isoforms is required to maintain the mitochondrial physiology in T. gondii tachyzoites.