Mol Genet Metab, 2004 Sep-Oct, 83(1-2), 28 - 37 Propionic acidemia: mutation update and functional and structural effects of the variant alleles; Desviat LR et al.; Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, a life-threatening inborn error of metabolism with autosomal recessive inheritance . To date, 41 mutations in the PCCA gene and 54 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of small insertions and deletions and splicing defects . A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with no prevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied . For the PCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations . These two populations show a different mutational spectrum, only sharing some involving CpG dinucleotides probably as recurrent mutational events . Functional characterization of the mutant missense alleles has been accomplished using different prokaryotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models . For the PCCA gene, the main molecular effect of the expressed mutations is related to protein instability, except two mutations in the active site predictably affecting ATP binding . In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site conformation resulting in diminished activity . A few carboxy-terminal PCCB mutations affect the interaction between subunits and the assembly with PCCA to form a functional PCC oligomer . The amount of normal transcripts resulting from some PCCA and PCCB splicing mutations has also been analyzed . Overall, the data generated from the expression analysis reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder. Parasitol Today, 1993 Apr, 9(4), 122 - 6 ATP versus pyrophosphate: glycolysis revisited in parasitic protists; Mertens E; It is generally assumed that glycolysis is remarkably similar among all organisms, prokaryotic and eukaryotic . This view has been extended to protists . However, there is growing evidence that significant deviations from conventional glycolysis occur in protists . Very different species, some parasitic, rely on peculiar enzymes that use pyrophosphate as substrate . In this review, Emmanuel Mertens describes such unusual pyrophosphate metabolism in parasitic protists. Parasitol Today, 1993 Oct, 9(10), 388 - 92 Genome analysis of Theileria parva; Morzaria SP et al.; Recent technological developments in the field of genome analyses have advanced our knowledge of the structures of prokaryotic and eukoryotic genomes . Examples of these range from small bacterial genomes, such as Escherichia coli, to the more complex genomes of Caenorhabditis elegans, Drosophila, humans and mouse . Here, Subhash Morzona and John Young review developments in mapping the genome of on economically important protozoan parasite o f cattle, Theileria parva . This map provides a framework for more detailed analysis of the genome structure o f this organism . The methodologies developed in constructing the map also have application to the mapping of other protozoan genomes. Parasitol Today, 1993 Oct, 9(10), 381 - 4 The Trypanosoma cruzi ribosomal P protein family: Classification and antigenicity; Levin MJ et al.; The multi-copy ribosomal P proteins have been identified on the ribosomes of prokaryotic and eukaryotic cells, and their antigenicity is an important feature of human Trypanosoma cruzi infection . In this review, Mariano Levin, Martin Vazquez, Dan Kaplan and Alejandro Schijman give a rational basis for the classification of these proteins, and discuss their inter-relationship. Parasitol Today, 1988 Dec, 4(12), 357 - 60 Polyamine biosynthesis and inhibition in Trichomonas vaginalis; Yarlett N; Polyomines - particularly putrescine, spermidine and spermine - are ubiquitous components of eukaryote and most prokaryote cells, and are essential for optimal cell proliferation . But since routes of polyamine synthesis may differ, for example between parasites and their hosts, selective inhibition of polyamine metabolism offers an attractive target for chemotherapy - as already shown with the success of difluoromethylomithine (DFMO) as an inhibitor of polyamine synthesis in African trypanosomes . Parasitology Today has featured a series of articles reviewing research on polyamine metabolism of various parasites (eg . vol . 3, pp 190-192, pp 312-315; vol . 4, pp 18-20) and here, Nigel Yorlett discusses these metabolic aspects of Trichomonas vaginalis (Fig . 1)-a common parasite of the urogenital tract. Parasitol Today, 1988 Jan, 4(1), 18 - 20 Polyamine metabolism of filaria and allied parasites; Walter RD; Putrescine and the polyamines spermidine and spermine occur both in prokaroytes and in eukaryotes where they seem intimately involved in regulatory processes of cellular growth and differentiation . They seem to play an important role related to the biosynthesis of nucleic acids and proteins, although at the molecular level their precise function remains unclear . In general, prokaryotes utilize putrescine and spermidine while eukaryotes tend to have higher concentrations of spermidine and spermine compared to putrescine(1-3.) Differences in polyamine metabolism between parasites and their hosts suggest several potential targets for chemotherapeutic attack As Rolf Walter discusses here, such approaches have already been exploited for African trypanosomes and also offer some leads for the chemotherapy of helminth infections. Acta Virol, 2004, 48(2), 97 - 107 Expression of herpes simplex virus 1 glycoprotein D in prokaryotic and eukaryotic cells; Mosko T et al.; Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed . The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells . The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain . The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein . The fusion protein was biotinylated and efficiently purified . The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis . In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed . This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages . The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.). J Mol Evol, 2004 Jun, 58(6), 712 - 21 Presequence acquisition during secondary endocytobiosis and the possible role of introns; Kilian O et al.; Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences . During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences . So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled . While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell . In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals . We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum . In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it . The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns. J Mol Evol, 2004 Jun, 58(6), 692 - 700 Cytosine methylation is not the major factor inducing CpG dinucleotide deficiency in bacterial genomes; Wang Y et al.; CpG dinucleotide deficiency has been found in viruses, mitochondria, prokaryotes, and eukaryotes . The consensual explanation is that it is due to deamination of methylated cytosines, as established for vertebrate and plants . However, we still do not know whether C5 cytosine methylation is also the major cause of CpG deficiency in bacteria . By combining annotation and experimental data identifying the presence of C5 cytosine methyltransferases with analysis of CpG relative abundance in 67 bacterial species, we found that CpG relative abundance in most bacterial genomes that have cytosine C5 methyltransferases tends to be in the normal range (observed/expected values between 0.82 and 1.21) . In contrast, many bacterial species likely to be lacking C5 cytosine methylation showed CpG deficiency . Furthermore, when comparing genomes with one another, TpG and CpA relative abundances were found to be independent from CpG relative abundance . This contrasted with intragenome analyses, where C3pG1 relative abundance (the subscripts refer to position of a nucleotide in a codon) was found to be generally positively correlated with T3pG1 relative abundances when plotted against GC content in protein coding sequences (CDSs) . This suggests the existence of alternative mechanisms contributing to CpG deficiency in bacteria. Nat Rev Mol Cell Biol, 2004 Oct, 5(10), 781 - 91 Pathways of chaperone-mediated protein folding in the cytosol; Young JC et al.; Cells are faced with the task of folding thousands of different polypeptides into a wide range of conformations . For many proteins, the folding process requires the action of molecular chaperones . In the cytosol of prokaryotic and eukaryotic cells, molecular chaperones of different structural classes form a network of pathways that can handle substrate polypeptides from the point of initial synthesis on ribosomes to the final stages of folding. J Microbiol, 2004 Sep, 42(3), 200 - 4 A proteomic approach to study msDNA function in Escherichia coli; Jeong MA et al.; Retron is a prokaryotic genetic element that produces multicopy single-stranded DNA covalently linked to RNA (msDNA) by a reverse transcriptase . It was found that cells producing a large amount of msDNA, rather than those that did not, showed a higher rate of mutation . In order to understand the molecular mechanism connecting msDNA production to the high mutation rate the protein patterns were compared by two dimensional gel electrophoresis . Ten proteins were found to be differentially expressed at levels more than three fold greater in cells with than without msDNA, nine of which were identified by MALDI TOF MS . Eight of the nine identified proteins were repressed in msDNA-producing cells and, surprisingly, most were proteins functioning in the dissimilation of various carbon sources . One protein was induced four fold greater in the msDNA producing cells and was identified as a 30S ribosomal protein S2 involved in the regulation of translation . The molecular mechanism underlying the elevated mutation in msDNA-producing cell still remains elusive . Nucleic Acids Res . 2004 Sep 29;32(17):e133. Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microarrays; Gordon PM et al.; We have developed a software package called Osprey for the calculation of optimal oligonucleotides for DNA sequencing and the creation of microarrays based on either PCR-products or directly spotted oligomers . It incorporates a novel use of position-specific scoring matrices, for the sensitive and specific identification of secondary binding sites anywhere in the target sequence . Using accelerated hardware is faster and more efficient than the traditional pairwise alignments used in most oligo-design software . Osprey consists of a module for target site selection based on user input, novel utilities for dealing with problematic sequences such as repeats, and a common code base for the identification of optimal oligonucleotides from the target list . Overall, these improvements provide a program that, without major increases in run time, reflects current DNA thermodynamics models, improves specificity and reduces the user's data preprocessing and parameterization requirements . Using a TimeLogic hardware accelerator, we report up to 50-fold reduction in search time versus a linear search strategy . Target sites may be derived from computer analysis of DNA sequence assemblies in the case of sequencing efforts, or genome or EST analysis in the case of microarray development in both prokaryotes and eukaryotes. Biochem J, 2005 Feb 1, 385(Pt 3), 703 - 13 The endo-beta-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours; Jam M et al.; Two beta-agarase genes, agaA and agaB, were functionally cloned from the marine bacterium Zobellia galactanivorans . The agaA and agaB genes encode proteins of 539 and 353 amino acids respectively, with theoretical masses of 60 and 40 kDa . These two beta-agarases feature homologous catalytic domains belonging to family GH-16 . However, AgaA displays a modular architecture, consisting of the catalytic domain (AgaAc) and two C-terminal domains of unknown function which are processed during secretion of the enzyme . In contrast, AgaB is composed of the catalytic module and a signal peptide similar to the N-terminal signature of prokaryotic lipoproteins, suggesting that this protein is anchored in the cytoplasmic membrane . Gel filtration and electrospray MS experiments demonstrate that AgaB is a dimer in solution, while AgaAc is a monomeric protein . AgaAc and AgaB were overexpressed in Escherichia coli and purified to homogeneity . Both enzymes cleave the beta-(1-->4) linkages of agarose in a random manner and with retention of the anomeric configuration . Although they behave similarly towards liquid agarose, AgaAc is more efficient than AgaB in the degradation of agarose gels . Given these organizational and catalytic differences, we propose that, reminiscent of the agarolytic system of Pseudoalteromonas atlantica, AgaA is specialized in the initial attack on solid-phase agarose, while AgaB is involved with the degradation of agarose fragments. Anal Chem, 2004 Oct 1, 76(19), 5930 - 6 Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutations in DNA; Palecek E et al.; MutS protein plays an important role in the DNA repair system in prokaryotic and eukaryotic cells; it recognizes unpaired and mispaired bases in duplex DNA and can be used for detection of point mutations in vitro . We have shown that small amounts of this protein can be detected electrochemically at mercury and carbon electrodes without any labeling . Using constant current stripping analysis (CPSA) and mercury electrodes, tens of attomoles of this protein can be detected . The sensitivity of the determination at carbon electrodes is by more than 3 orders of magnitude lower . Using biotinylated DNA duplexes attached to magnetic beads, single-base mismatches and insertion/deletions were recognized by MutS . Picogram amounts of this protein were detected by CPSA after MutS releasing from the beads. J Med Chem, 2004 Oct 7, 47(21), 5149 - 58 Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates; Cheng F et al.; We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock . The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically . The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008) . The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues . Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme . The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH . Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E . coli enzyme) . Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes. FEMS Microbiol Lett, 2004 Oct 1, 239(1), 117 - 23 Abundance of 4Fe-4S motifs in the genomes of methanogens and other prokaryotes; Major TA et al.; The abundance of 4Fe-4S motifs of the form CX2CX2CX3C was analyzed in the open reading frames (ORFs) of 120 prokaryotic genomes . The abundance of ORFs containing the CX2CX2CX3C motif or isORFs correlated (r=0.82) with methanogenesis (p=0.0001), archaea (p=0.0173), anaerobiosis (p<0.0001) and genome size (p<0.0001) . Optimal growth temperature (hyperthermophily) did not correlate with the number of isORFs (p=0.6283) . Large numbers of CX2CX2CX3C motifs may be associated with unique physiologies: methanogenic archaea contained the greatest number of CX2CX2CX3C motifs found among the prokaryotic groups; however, only about 15% of the motifs were in genes directly involved in methanogenesis . Large numbers of CX2CX2CX3C motifs may also be associated with generalists such as Desulfitobacterium hafniense, which is an anaerobic bacterium containing multiple reductases. Dev Comp Immunol, 2005, 29(2), 143 - 52 Cloning and characterization of chicken stromal cell derived factor-1; Read LR et al.; Stromal cell derived factor-1, SDF-1, belongs to the CXC family of chemokines and has been identified in mammals, amphibians, and fish . This chemokine has a diverse array of functions in organogenesis, hematopoeisis, B cell development and recruitment of immune system cells . Here, we report the cloning of the chicken SDF-1 ortholog and examine its temporal and spatial expression . The chicken SDF-1 cDNA contained an open reading frame encoding a predicted protein of 89 amino acids, which shared 40-75% identity to SDF-1 protein in other species . Protein folding simulation predicted a tertiary structure very similar to that obtained for human SDF-1 . Recombinant chicken SDF-1 was produced using a prokaryotic expression system and the recombinant protein was shown to be biologically active in a calcium flux assay . The SDF-1 gene was found to be expressed ubiquitously and constitutively in adult tissues and was present as early as the primitive streak stage of chicken embryos. Trends Biochem Sci, 2004 Oct, 29(10), 535 - 41 A common folding mechanism in the cytochrome c family; Travaglini-Allocatelli C et al.; Of the globular proteins, cytochrome c (cyt c) has been used extensively as a model system for folding studies . Here we analyse the folding pathway of different cyt c proteins from prokaryotes and eukaryotes, and attempt to single out general correlations between structural determinants and folding mechanisms . Recent studies provide evidence that the folding pathway of several cyt c proteins involves the formation of a partially structured intermediate . Using state-of-the-art kinetic analysis on published data, we show that such a folding intermediate is an obligatory on-pathway species that might represent either a defined local minimum in the reaction coordinate or an unstable high-energy state . Available data also indicate that some essential structural features of the folding intermediate and transition states are highly conserved across this protein family . Thus, cyt c proteins share a consensus folding mechanism in spite of large differences in physico-chemical properties and thermodynamic stability . This novel outlook on the folding of cyt c can shed light on much published data and might offer a general scheme by which to plan new experiments. Biol Chem, 2004 Aug, 385(8), 749 - 54 Distinctive functional features in prokaryotic and eukaryotic Cu,Zn superoxide dismutases; Gabbianelli R et al.; Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization . We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme . Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site . Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3- -dependent peroxidative activity . Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E . coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K . We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility. Mol Vis, 2004 Sep 08, 10, 655 - 62 The IXI/V motif in the C-terminal extension of alpha-crystallins: alternative interactions and oligomeric assemblies; Pasta SY et al.; PURPOSE: Alpha-crystallin, a hetero-oligomer of alphaA- and alphaB-crystallin, is involved in maintaining eye lens transparency, primarily by its structural packing and chaperone activity . alphaA- and alphaB-crystallin share significant sequence homology, which is almost exclusively restricted to the central, conserved "alphaA-crystallin domain" . The flanking N-terminal domain and C-terminal extension are highly variable both in sequence and length . Mutations and age-related post-translational modifications of these proteins are associated with congenital and age-onset cataracts . Interestingly, most mutations or truncations in the C-terminal extensions of the alpha-crystallins and other alpha-sHsps like Hsp27 lead to pathology . It is therefore important to understand the structure/function relationship of this region . Sequence alignment of the C-terminal extensions of alphaA- and alphaB-crystallin with other homologues shows a conserved IXI/V motif . The purpose of this study was to investigate the role of this conserved motif, IPV in alphaA-crystallin and IPI in alphaB-crystallin (corresponding to residues 159-161 in both crystallins), in the structure and chaperone activity . METHODS: The isoleucine/valine residues in the IPV motif of alphaA-crystallin and the IPI motif of alphaB-crystallin were mutated to glycine and studied the secondary and tertiary structure of the mutant proteins using circular dichroism and fluorescence spectroscopy, and the quaternary structure using glycerol density gradient centrifugation and dynamic light scattering . Chaperone activity was studied at 37 degrees C and 25 degrees C using DTT induced aggregation of insulin as a model system . We have performed fluorescence resonance energy transfer (FRET) experiments to investigate the interactions of this motif in homo- and hetero-oligomers . Since alphaB-crystallin is devoid of Cys residues, we have introduced a Cys residue flanking the IPI motif (T162CalphaB-crystallin) to facilitate fluorescence labeling studies . RESULTS: Unlike in other homologues from plants or prokaryotes, mutation of the isoleucine/valine residues in alpha-crystallins does not result in oligomer dissociation or loss of chaperone activity . On the contrary, the mutant proteins retain their capacity to oligomerize and show enhanced chaperone activity at 37 degrees C . The mutants also exhibit significantly higher chaperone-like activity at 25 degrees C . FRET experiments show that the region spanning the IPI/V motif comes in proximity either to the beta-strands of the "alpha-crystallin" domain or the corresponding IPI/V region of another subunit . CONCLUSIONS: Our mutational studies show that the IPI/V motif has a propensity to participate in inter-subunit interactions, either with regions in the alpha-crystallin domain or with the corresponding IPI/V region on another monomer . These interactions are important in the structure and function of alpha-crystallins . This motif also appears to be important in the temperature dependent chaperone-like activity of the alpha-crystallins . The propensity of the IPI/V motif to form multiple inter-subunit interactions may contribute to the diversity in structure and function seen in the alpha-crystallin/sHsp family. Nucleic Acids Res, 2004 Sep 24, 32(17), 5036 - 44 Print 2004. Solving the riddle of codon usage preferences: a test for translational selection; dos Reis M et al.; Translational selection is responsible for the unequal usage of synonymous codons in protein coding genes in a wide variety of organisms . It is one of the most subtle and pervasive forces of molecular evolution, yet, establishing the underlying causes for its idiosyncratic behaviour across living kingdoms has proven elusive to researchers over the past 20 years . In this study, a statistical model for measuring translational selection in any given genome is developed, and the test is applied to 126 fully sequenced genomes, ranging from archaea to eukaryotes . It is shown that tRNA gene redundancy and genome size are interacting forces that ultimately determine the action of translational selection, and that an optimal genome size exists for which this kind of selection is maximal . Accordingly, genome size also presents upper and lower boundaries beyond which selection on codon usage is not possible . We propose a model where the coevolution of genome size and tRNA genes explains the observed patterns in translational selection in all living organisms . This model finally unifies our understanding of codon usage across prokaryotes and eukaryotes . Helicobacter pylori, Saccharomyces cerevisiae and Homo sapiens are codon usage paradigms that can be better understood under the proposed model. J Biol Chem, 2004 Nov 5, 279(45), 47076 - 80 Epub 2004 Sep 23. Functional characterization of a prokaryotic Kir channel; Enkvetchakul D et al.; The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells . A related gene family (KirBac) has recently been identified in prokaryotes . While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products . Here we present functional characterization of KirBac1.1 reconstituted in liposomes . Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions . In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6) . This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel. Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1895 - 902 A proposal for further integration of the cyanobacteria under the Bacteriological Code; Oren A; This taxonomic note reviews the present status of the nomenclature of the cyanobacteria under the Bacteriological Code . No more than 13 names of cyanobacterial species have been proposed so far in the International Journal of Systematic and Evolutionary Microbiology (IJSEM)/International Journal of Systematic Bacteriology (IJSB), and of these only five are validly published . The cyanobacteria (Cyanophyta, blue-green algae) are also named under the Botanical Code, and the dual nomenclature system causes considerable confusion . This note calls for a more intense involvement of the International Committee on Systematics of Prokaryotes (ICSP), its Judicial Commission and its Subcommittee on the Taxonomy of Photosynthetic Prokaryotes in the nomenclature of the cyanobacteria under the Bacteriological Code . The establishment of minimal standards for the description of new species and genera should be encouraged in a way that will be acceptable to the botanical authorities as well . This should be followed by the publication of an 'Approved List of Names of Cyanobacteria' in IJSEM . The ultimate goal is to achieve a consensus nomenclature that is acceptable both to bacteriologists and to botanists, anticipating the future implementation of a universal 'Biocode' that would regulate the nomenclature of all organisms living on Earth. Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1427 - 8 Notification that new names and new combinations have appeared in volume 54, part 3, of the IJSEM; Cyanobacterial genes transmitted to the nucleus before divergence of red algae in the Chromista; Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan . nozaki@biol.s.u-tokyo.ac.jp The plastids of red algae, green plants, and glaucophytes may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis . In contrast, the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events involving a phototrophic eukaryote and a eukaryotic host cell . Although phylogenetic analyses of multiple plastid genes from a wide range of eukaryotic lineages have been carried out, the phylogenetic positions of the secondary plastids of the Chromista (Heterokontophyta, Haptophyta and Cryptophyta) are ambiguous in a range of different analyses . This ambiguity may be the result of unusual substitutions or bias in the plastid genes established by the secondary endosymbiosis . In this study, we carried out phylogenetic analyses of five nuclear genes of cyanobacterial origin (6-phosphogluconate dehydrogenase {gnd}, oxygen-evolving-enhancer {psbO}, phosphoglycerate kinase {pgk}, delta-aminolevulinic acid dehydratase {aladh}, and ATP synthase gamma {atpC} genes), using the genome sequence data from the primitive red alga Cyanidioschyzon merolae 10D . The sequence data robustly resolved the origin of the cyanobacterial genes in the nuclei of the Chromista (Heterokontophyta and Haptophyta) and Dinophyta, before the divergence of the extant red algae (including Porphyra {Rhodophyceae} and Cyanidioschyzon {Cyadidiophyceae}) . Although it is likely that gnd genes in the Chromista were transmitted from the cyanobacterium-like ancestor of plastids in the primary endosymbiosis, other genes might have been transferred from nuclei of a red algal ancestor in the secondary endosymbiosis . Therefore, the results indicate that the Chromista might have originated from the ancient secondary endosymbiosis before the divergence of extant red algae. J Mol Evol, 2004 Jul, 59(1), 59 - 71 Comparative analysis of the ribosomal components of the hydrogenosome-containing protist, Trichomonas vaginalis; Arisue N et al.; The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes . In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized . The sedimentation coefficient of the T . vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli . Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80 . This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E . coli (approximately 55) . N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features . Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T . vaginalis sequences are positioned within a eukaryotic clade . Comparison of deduced secondary structure models of the small and large subunit rRNAs of T . vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T . vaginalis, while variable regions are shortened or lost . These lines of evidence demonstrate that the T . vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type. J Mol Evol, 2004 Jul, 59(1), 51 - 8 New aspects on lanosterol 14alpha-demethylase and cytochrome P450 evolution: lanosterol/cycloartenol diversification and lateral transfer; Rezen T et al.; Sterol 14alpha-demethylase (CYP51) is a member of the cytochrome P450 superfamily, widely found in animals, fungi, and plants but present in few prokaryotic groups . CYP51 is currently believed to be the ancestral cytochrome P450 that has been transferred from prokaryotes to eukaryotic kingdoms . We propose an alternate view of CYP51 evolution that has an impact on understanding the evolution of the entire CYP superfamily . Two hundred forty-nine bacterial and four archaeal CYP sequences have been aligned and a bacterial CYP tree designed, showing a separation of two branches . Prokaryotic CYP51s cluster to the minor branch, together with other eukaryote-like CYPs . Mycobacterial and methylococcal CYP51s cluster together (100% bootstrap probability), while Streptomyces CYP51 remains on a distant branch . A CYP51 phylogenetic tree has been constructed from 44 sequences resulting in a ((plant, bacteria),(animal, fungi)) topology (100% bootstrap probability) . This is in accordance with the lanosterol/cycloartenol diversification of sterol biosynthesis . The lanosterol branch (nonphotosynthetic lineage) follows the previously proposed topology of animal and fungal orthologues (100% bootstrap probability), while plant and D . discoideum CYP51s belong to the cycloartenol branch (photosynthetic lineage), all in accordance with biochemical data . Bacterial CYP51s cluster within the cycloartenol branch (69% bootstrap probability), which is indicative of a lateral gene transfer of a plant CYP51 to the methylococcal/mycobacterial progenitor, suggesting further that bacterial CYP51s are not the oldest CYP genes . Lateral gene transfer is likely far more important than hitherto thought in the development of the diversified CYP superfamily . Consequently, bacterial CYPs may represent a mixture of genes with prokaryotic and eukaryotic origin. IEEE Trans Nanobioscience, 2003 Mar, 2(1), 29 - 34 Parallel pattern identification in biological sequences on clusters; Huang CH et al.; Tandem repeats are ubiquitous sequence features in both prokaryotic and eukaryotic genomes . They are known to cause several inherited neurological diseases in humans . Identifying these patterns is a highly computation-intensive process . Previous parallel implementations use straightforward domain decomposition based on existing sequential algorithms and rely on parallel machines with low-latency interconnection network and fast hardware support for processor synchronization . Our research exploits the superior cost effectiveness and flexibility achieved through low-cost clusters to speed up biological computations by designing communication-efficient parallel algorithms for pattern identification . This paper presents a low communication-overhead parallel algorithm for pattern identification in biological sequences . Given a biological sequence of length n and a pattern of length m, we conclude an algorithm with five computation/communication phases, each requiring O(n) computation time and only O(p) message units . The low communication overhead of the algorithm is essential in achieving reasonable speedups on clusters, where the inter-processor communication latency is usually higher. Physiology (Bethesda), 2004 Oct, 19, 293 - 9 A two-holed story: structural secrets about ClC proteins become unraveled? Babini E, Pusch M. ClC Cl(-) channels are found in almost all organisms, ranging from bacteria to mammals, in which nine Cl(-) channels belonging to the ClC family have been identified . The biophysical properties and physiological functions of ClC Cl(-) channels have been extensively reviewed . In this short review, we will focus on recent results obtained on the X-ray structure and functional properties of the prokaryotic ClC-ec1 protein and some results obtained on the role of the cytoplasmic COOH terminus of mammalian ClCs. J Mol Biol, 2004 Oct 8, 343(1), 249 - 65 Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems; Garcia O et al.; ATP-binding cassette (ABC) proteins constitute a large superfamily found in all kingdoms of living organisms . The recent completion of two draft sequences of the rice (Oryza sativa) genome allowed us to analyze and classify its ABC proteins and to compare to those in Arabidopsis thaliana . We identified a similar number of ABC proteins in rice and Arabidopsis (121 versus 120), despite the rice genome being more than three times the size of Arabidopsis . Both Arabidopsis and rice have representative members in all seven major subfamilies of ABC ATPases (A to G) commonly found in eukaryotes . This comparative analysis allowed the detection of 29 potential orthologous sequences in Arabidopsis and rice . However, plant share with prokaryotes a specific set of ABC systems that is not detected in animals . These ABC systems might be inherited from the cyanobacterial ancestor of chloroplasts . The present work provides the first complete inventory of rice ABC proteins and an updated inventory of those proteins in Arabidopsis. J Mol Biol, 2004 Oct 8, 343(1), 1 - 28 STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer; Leipe DD et al.; Using sequence profile analysis and sequence-based structure predictions, we define a previously unrecognized, widespread class of P-loop NTPases . The signal transduction ATPases with numerous domains (STAND) class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g . NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria . We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families . We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins . The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases . The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain . This helical module contains a signature GxP motif in the loop between the two distal helices . With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains . In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases . By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains . STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide . The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer . The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes . Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems. Int J Biochem Cell Biol, 2005 Jan, 37(1), 54 - 68 TB tools to tell the tale-molecular genetic methods for mycobacterial research; Machowski EE et al.; In spite of the availability of drugs and a vaccine, tuberculosis--one of man's medical nemeses--remains a formidable public health problem, particularly in the developing world . The persistent nature of the tubercle bacillus, with one third of the world's population is estimated to be infected, combined with the emergence of multi drug-resistant strains and the exquisite susceptibility of HIV-positive individuals, has underscored the urgent need for in-depth study of the biology of Mycobacterium tuberculosis address the resurgence of TB . In aiming to understand the mechanisms by which mycobacteria react to their immediate environments, molecular genetic tools have been developed from naturally occurring genetic elements . These include protein expressing genes, and episomal and integrating elements, which have been derived mainly from prokaryotic but also from eukaryotic organisms . Molecular genetic tools that had been established as routine procedures in other prokaryotic genera were thus mimicked . Knowledge of the underlying mechanisms greatly expedited the harnessing of these elements for mycobacteriological research and has brought us to a point where these molecular genetic tools are now employed routinely in laboratories worldwide. FEMS Yeast Res, 2004 Oct, 5(1), 11 - 8 Therapeutic potential of yeast killer toxin-like antibodies and mimotopes; Magliani W et al.; This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy . KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms . They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats . KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics. Curr Biol, 2004 Sep 21, 14(18), R768 - 70 How do prokaryotic cells cycle? Margolin W, Bernander R. This issue of Current Biology features five reviews covering various key aspects of the eukaryotic cell cycle . The topics include initiation of chromosome replication, assembly of the mitotic spindle, cytokinesis, the regulation of cell-cycle progression, and cell-cycle modeling, focusing mainly on budding yeast, fission yeast and animal cell model systems . The reviews underscore common themes as well as key differences in the way these processes are carried out and regulated among the different model organisms . Consequently, an important question is how cell-cycle mechanisms and controls have evolved, particularly in the broader perspective of the three domains of life. Mini Rev Med Chem, 2004 Sep, 4(7), 721 - 39 The relationship between inhibitors of eukaryotic and prokaryotic serine proteases; Konaklieva MI et al.; The ability to inhibit serine proteases is a major focus in the pharmaceutical industry . Serine proteases of medical importance range in phylogenetic diversity from the metallo-proteases, which play a role in pulmonary hypertension, and destruction of the lung parenchyma in emphysema, to those proteases (beta-lactamases), which play a role in the resistance of bacteria to beta-lactam antibiotics . In both the mammalian and microbial systems, the development of serine protease inhibitors has been a focal strategy spurring investigations in the area of serine protease dependent prodrugs that incorporate a bactericidal moiety as well as other classes of metalloprotease inhibitors. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2004 Aug, 26(4), 368 - 71 {Clone and expression of isocitrate lyase gene in Mycobacterium tuberculosis H37Rv}; Li DW et al.; OBJECTIVE: To construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system . METHODS: The recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning . The fusion protein was expressed in E . coli host strain BL21 (DE3) . Activity of the fusion protein was studied after it was purified with metal chelating chromatography . RESULTS: We constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL . The plasmid was highly expressed in E . coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content . After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90% . The fusion protein had activity of ICL . CONCLUSION: Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point. Nat Rev Microbiol, 2004 Oct, 2(10), 802 - 8 Chlamydia and apoptosis: life and death decisions of an intracellular pathogen; Byrne GI et al.; The chlamydiae are important obligate intracellular prokaryotic pathogens that, each year, are responsible for millions of human infections involving the eye, genital tract, respiratory tract, vasculature and joints . The chlamydiae grow in cytoplasmic vesicles in susceptible host cells, which include the mucosal epithelium, vascular endothelium, smooth muscle cells, circulating monocytes and recruited or tissue-specific macrophages . One important pathogenic strategy that chlamydiae have evolved to promote their survival is the modulation of programmed cell death pathways in infected host cells . The chlamydiae can elicit the induction of host cell death, or apoptosis, under some circumstances and actively inhibit apoptosis under others . This subtle pathogenic mechanism highlights the manner in which these highly successful pathogens take control of infected cells to promote their own survival - even under the most adverse circumstances. Bioinformatics . 2004 Sep 16; {Epub ahead of print} Improving genome annotations using phylogenetic profile anomaly detection; Mikkelsen TS et al.; MOTIVATION: A promising strategy for refining genome annotations is to detect features that conflict with known functional or evolutionary relationships between groups of genes . Previous work in this area has been focused on investigating the absence of "housekeeping" genes or components of well-studied pathways . We have sought to develop a method for improving new annotations that can automatically synthesize and use the information available in a database of other annotated genomes . RESULTS: We show that a probabilistic model of phylogenetic profiles, trained from a database of curated genome annotations, can be used to reliably detect errors in new annotations . We use our method to identify 22 genes that were missed in previously published annotations of prokaryotic genomes . AVAILABILITY: The method was evaluated using MATLAB and open source software referenced in this work . Scripts and datasets are available from the authors upon request. Arch Gerontol Geriatr, 1990 Mar-Apr, 10(2), 207 - 16 'Oxygen toxicity' induced by isoproterenol oxidation on living cells . An in vitro prokaryotic model; Giunta S et al.; Oxygen radical damage is a relevant problem in gerontological research . It has been implicated both in the aging process itself and in aging-related pathologies . Oxygen radicals from catecholamines seem to play an important role in central nervous system and cardiovascular system disorders during aging . Prokaryotic experimental systems have been shown to provide a simple and short term in vitro model for 'oxygen toxicity' from catecholamine oxidation which might be useful also in age-related research . In this paper we show that the synthetic sympathomimetic catecholamine, isoproterenol, oxidizes when added to bacteriological media and that this oxidation process causes bacterial growth inhibition . Both isoproterenol oxidation and the growth-inhibitory activity can be prevented by the addition of antioxidants . The addition of exogenous catalase (CAT), while unable to prevent isoproterenol oxidation, totally suppressed the bacterial growth inhibition; the addition of exogenous superoxide dismutase partially antagonized isoproterenol oxidation and suppressed bacterial growth inhibition although less efficiently than CAT . The model described suggests that besides 'oxygen toxicity' by endogenous natural catecholamines, iatrogenic tissue injury caused by the oxidation intermediates from this class of pharmacological compounds must also be considered. J Biol Chem, 2004 Nov 12, 279(46), 47906 - 11 Epub 2004 Sep 14. Lipopolysaccharide-free heat shock protein 60 activates T cells; Osterloh A et al.; A possible function of eukaryotic heat shock protein 60 (Hsp60) as endogenous danger signal has been controversially discussed in the past . Hsp60 was shown to induce the secretion of proinflammatory cytokines in professional antigen-presenting cells and to enhance the activation of T cells in primary stimulation . However, in vitro activation of macrophages by Hsp60 was attributed to contaminating endotoxin in the recombinant Hsp60 protein preparations . Here, we employ low endotoxin recombinant human Hsp60 and murine Hsp60 expressed by eukaryotic cell lines to dissect the Hsp60 protein-mediated effects from biologic effects that are mediated by prokaryotic contaminants in the Hsp60 protein preparation . The induction of tumor necrosis factor-alpha secretion in mouse macrophages is lost after endotoxin removal and is not mediated by Hsp60 expressed in eukaryotic systems . In contrast, the Hsp60-mediated enhancement of antigen-specific T cell activation does not correlate with endotoxin contamination . Moreover, Hsp60 that is expressed on the surface of different eukaryotic cell lines increases the activation of T cells in primary stimulation . Taken together, we provide evidence that endogenous Hsp60, which is thought to be released from dying infected cells in vivo, has a biological function that is not due to contaminating pathogen-associated molecules. J Biol Chem, 2004 Nov 26, 279(48), 50437 - 45 Epub 2004 Nov 26. Phage phi29 DNA replication organizer membrane protein p16.7 contains a coiled coil and a dimeric, homeodomain-related, functional domain; Munoz-Espin D et al.; The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in prokaryotic membrane-associated DNA replication . Protein p16.7 contains an N-terminal transmembrane domain responsible for membrane localization . A soluble variant lacking the N-terminal membrane anchor, p16.7A, forms dimers in solution, binds to DNA, and has affinity for the varphi29 terminal protein . Here we show that the soluble N-terminal half of p16.7A can form a dimeric coiled coil . However, a second domain, located in the C-terminal half of the protein, has been characterized as being the main domain responsible for p16.7 dimerization . This 70-residue C-terminal domain, named p16.7C, also constitutes the functional part of the protein as it binds to DNA and terminal protein . Sequence alignments, secondary structure predictions, and spectroscopic analyses suggest that p16.7C is evolutionarily related to DNA binding homeodomains, present in many eukaryotic transcriptional regulator proteins . Based on the results, a structural model of p16.7 is presented. Genes Dev, 2004 Sep 15, 18(18), 2183 - 94 The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease; Giaccia AJ et al.; The ability to sense and respond to changes in oxygen is essential for the survival of prokaryotic and eukaryotic organisms . Oxygen-sensing mechanisms have been developed to maintain cell and tissue homeostasis, as well as to adapt to the chronic low-oxygen conditions found in diseases such as cancer . This report on the first Keystone Meeting on the Biology of Hypoxia will summarize our current understanding of key genes and pathways involved in oxygen sensing that are required for normal development and that are dysregulated in disease states . It will also comment on future directions for this exciting field. In Vivo, 2004 Jul-Aug, 18(4), 505 - 7 Effect of a trifluoromethyl ketone on the motility of proton pump-deleted mutant of Escherichia coli strain and its wild-type; Molnar A et al.; We have recently found that 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone {TF18} exhibited the most potent antibacterial activity among 30 trifluoromethyl ketones against various prokaryotes, such as Escherichia coli (E . coli) . In the present study, the inhibition of E . coli motility by TF18 was investigated . TF18 showed the lowest minimum inhibitory concentration (MIC) and highest inhibitory effect on the motility of E . coli strains . The wild-type E . coli was more sensitive to inhibition of motility than its proton pump-deleted mutant strain at subinhibitory concentrations . These data suggest that one of the targets of the antibacterial effect of the trifluoromethyl ketone is the proton pump system. Plant Cell, 2004 Oct, 16(10), 2665 - 82 Epub 2004 Sep 14. DNA gyrase is involved in chloroplast nucleoid partitioning; Cho HS et al.; DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes . Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana . NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar . NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles . Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles . Furthermore, 4',6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma . Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant . Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Sep, 20(5), 578 - 81 {Expression of GST-PRL-2 fusion protein in prokaryotic cells and preparation of Hen egg yolk immunoglobulin (IgY) against PRL-2}; Guo AL et al.; AIM: To express phosphatase 2 of regenerating liver (PRL-2) fusion protein in E.coli and to prepare specific hen egg yolk immunoglobulin(IgY) against PRL-2 . METHODS: A full-length human PRL-2 gene coding sequence was cloned into expression vectors pGEX-4T-2 and pET21a, then transformed into E.coli . Fusion protein GST-PRL-2 was expressed in E.coli via IPTG induction . The expressed proteins were purified from lysates through Glutathione Sepharose 4B and the Ni-NTA agarose columns, respectively . Egg-laying hens were immunized using the purified GST-PRL-2 with Freund's complete or incomplete adjuvant . The specificity of the resulting antibody was identified by Western blot . RESULTS: A high level of expression of target protein was detected by Western blot after IPTG induction and purified protein was obtained through Glutathione Sepharose 4B and the Ni-NTA affinity chromatography agarose columns, respectively . Western blot analysis showed that the anti-PRL-2 polyclonal antibody can recognize 6xHis-PRL-2 fusion protein . CONCLUSION: The hen egg yolk immunoglobulin(IgY) against PRL-2 expressed in E.coli has good specificity which provides an useful reagent for the detection of PRL-2. Int Rev Cytol, 2004, 238, 59 - 118 Organelle nuclei in higher plants: structure, composition, function, and evolution; Sakai A et al.; Plant cells have two distinct types of energy-converting organelles: plastids and mitochondria . These organelles have their own DNAs and are regarded as descendants of endosymbiotic prokaryotes . The organelle DNAs associate with various proteins to form compact DNA-protein complexes, which are referred to as organelle nuclei or nucleoids . Various functions of organelle genomes, such as DNA replication and transcription, are performed within these compact structures . Fluorescence microscopy using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole has played a pivotal role in establishing the concept of "organelle nuclei." This fluorochrome has also facilitated the isolation of morphologically intact organelle nuclei, which is indispensable for understanding their structure and composition . Moreover, development of an in vitro transcription?DNA synthesis system using isolated organelle nuclei has provided us with a means of measuring and analyzing the function of organelle nuclei . In addition to these morphological and biochemical approaches, genomics has also had a great impact on our ability to investigate the components of organelle nuclei . These analyses have revealed that organelle nuclei are not a vestige of the bacterial counterpart, but rather are a complex system established through extensive interaction between organelle and cell nuclear genomes during evolution . Extensive diversion or exchange during evolution is predicted to have occurred for several important structural proteins, such as major DNA-compacting proteins, and functional proteins, such as RNA and DNA polymerases, resulting in complex mechanisms to control the function of organelle genomes . Thus, organelle nuclei represent the most dynamic front of interaction between the three genomes (cell nuclear, plastid, and mitochondrial) constituting eukaryotic plant cells. Gene, 2004 Sep 15, 339, 149 - 60 The origin and evolution of eucaryal HIS7 genes: from metabolon to bifunctional proteins? Brilli M, Fani R. The fifth step of histidine biosynthesis is catalysed by an imidazole glycerol-phosphate (IGP) synthase . In Archaea and Bacteria, the active form of IGP synthase is a stable 1:1 dimeric complex constituted by a glutamine amidotransferase (GAT) and a cyclase, the products of hisH and hisF . In Eucarya, the two activities are associated with a single bifunctional polypeptide encoded by HIS7 . In this work, we report a comparative analysis of the amino acid sequence of all the available HisH, HisF and HIS7 proteins, which allowed depicting a likely evolutionary pathway leading to the present-day bifunctional HIS7 genes . According to the model that we propose, the bifunctional HIS7 gene is the outcome of a gene fusion event between two independent ancestral cistrons encoding an amidotransferase and a cyclase, respectively . The phylogenetic distribution of the eucaryal HIS7 genes and the analysis of all the available prokaryotic counterparts (hisH and hisF) revealed the absence of such fusions in prokaryotes, suggesting that the fusion event very likely occurred in an early stage of eucaryal evolution and was fixed in the nucleated cells . The biological significance of this gene fusion is also discussed. Trends Parasitol, 2004 Oct, 20(10), 462 - 8 Genomic filtering: an approach to discovering novel antiparasitics; McCarter JP; Genomic filtering is a rapid approach to identifying and prioritizing molecular targets for drug discovery . For infectious disease applications, comparative genomics filters allow the selection of pathogen-specific gene products, whereas functional genomics filters, such as RNA interference (RNAi), allow the selection of gene products essential for pathogen survival . The approach is especially applicable to antiparasitic drug discovery where the phylogenetic distance between parasite and host make the likelihood of drug cross-toxicity due to conservation of molecular targets greater than for more distantly related pathogens such as prokaryotes . This article discusses some of the inherent challenges of applying genomics to the early steps of drug discovery and describes one successful comparative and functional genomics filtering strategy that has been implemented to prioritize molecular targets and identify chemical leads for nematode control. PLoS Biol . 2004 Sep;2(9):E277 . Epub 2004 Sep 07. A Drosophila pattern recognition receptor contains a peptidoglycan docking groove and unusual L,D-carboxypeptidase activity; Chang CI et al.; The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes . We have determined the crystal structure of PGRP-SA to 2.2-A resolution and analyzed its peptidoglycan (PG) recognition and signaling activities . We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs . Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation . Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity . As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG. J Biol Chem, 2004 Nov 26, 279(48), 49656 - 63 Epub 2004 Nov 26. Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons; Tang HL et al.; Although initiation of translation at non-AUG codons occurs occasionally in prokaryotes and higher eukaryotes, it has not been reported in yeast until very recently . Evidence presented here shows that redundant ACG codons are recognized as alternative translation start sites for ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase . ALA1 is shown to be a bifunctional gene that provides both cytoplasmic and mitochondrial activities . Unlike most bifunctional genes that contain alternative in-frame AUG initiators, there is only one AUG codon, designated AUG1, close to the 5'-end of the ALA1 open reading frame . Transcriptional mapping identified three overlapping transcripts, with 5'-ends at positions 54, 105, and 117 nucleotides upstream of AUG1, respectively . Site-specific mutagenesis demonstrated that the cytoplasmic and mitochondrial functions of ALA1 are provided by two protein isoforms with distinct amino termini; that is, a short cytoplasmic form initiated at AUG1 and a longer mitochondrial isoform initiated at two upstream in-frame ACG codons, i.e . ACG(-25) and ACG(-24) . These two ACG codons function redundantly in initiation of translation . Either codon can function in the absence of the other . The short transcript appears to serve as the template for the cytoplasmic form, whereas the longer transcripts are likely to code for both isoforms via alternative initiation . Because yeast ribosomes in general cannot efficiently recognize a non-AUG initiator, this unique feature of redundancy of non-AUG initiators in a single mRNA may in itself represent a novel paradigm for translation initiation from poor initiators. FEBS Lett, 2004 Sep 10, 574(1-3), 101 - 5 A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions; Hihara Y et al.; Pirin is a recently identified protein in eukaryotes as a transcription cofactor or as an apoptosis-related protein . Although Pirin is highly conserved from bacteria to human, there have been no reports on prokaryotic Pirin orthologs . We show here that pirA (sll1773) encoding an ortholog of Pirin together with an adjacent gene, pirB (ssl3389), was upregulated under high salinity and some other stress conditions in a cyanobacterium Synechocystis sp . PCC 6803 . Induction of the pirAB genes was not related to cell death and disruption of pirA did not affect the gene expression profile . Expression of the pirAB genes was negatively regulated by a LysR family transcriptional regulator encoded by pirR (slr1871) located immediately upstream of pirAB in the divergent direction . DNA microarray analysis indicated that PirR repressed expression of closely located ORFs, slr1870 and mutS (sll1772), in addition to pirAB and pirR itself. Trends Plant Sci, 2004 Aug, 9(8), 371 - 7 The ALDH gene superfamily of Arabidopsis; Kirch HH et al.; Aldehyde dehydrogenases (ALDHs) represent a protein superfamily of NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes . The Arabidopsis genome contains 14 unique ALDH sequences encoding members of nine ALDH families, including eight known families and one novel family (ALDH22) that is currently known only in plants . Here, we identify members of the ALDH gene superfamily in Arabidopsis; provide a revised, unified nomenclature for these ALDH genes; analyze the molecular relationship among Arabidopsis ALDH genes and compare them to ALDH genes from other species, including prokaryotes and mammals; and describe the role of ALDHs in cytoplasmic male sterility, plant defense and abiotic stress tolerance. BMC Bioinformatics . 2004 Sep 09;5(1):127. Functionally specified protein signatures distinctive for each of the different blue copper proteins; Giri AV et al.; BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint . The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences . Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes . The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests . Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families . This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins . The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands {each from histidine} and one sulphur containing thiolate ligand {from cysteine}, with strong interactions between the copper center and these ligands . RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership . The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes . ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides . The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom . It was highly specific for each kind of blue copper protein and the false picks were minimized . The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section . CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences. Plant Cell Physiol, 2004 Aug, 45(8), 960 - 7 ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase; Shimada H et al.; The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division . To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division . Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes . The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif . Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division . Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis. Mol Biol Evol, 2005 Jan, 22(1), 21 - 28 Epub 2004 Sep 8. Unique Origin and Lateral Transfer of Prokaryotic Chlorophyll-b and Chlorophyll-d Light-Harvesting Systems; Chen M et al.; pcb genes, encoding proteins binding light-harvesting chlorophylls, were cloned and sequenced from the Chl d-containing cyanobacterium, Acaryochloris marina, and the Chl b-containing cyanobacterium, Prochloron didemni . Both organisms contained two tandem pcb genes . Peptide fingerprinting confirmed the expression of one of the A . marina pcb genes . Phylogenetic tree reconstruction using distance-matrix and maximum-likelihood methods indicated a single origin of the pcb gene family, whether occurring in Chl b-containing or Chl d-containing organisms . This may indicate widespread lateral transfer of the Pcb protein-based light-harvesting system. Annu Rev Cell Dev Biol . 2004 Jul 2; {Epub ahead of print} Cellular Length Control Systems; Marshall WF; The problem of organelle size control can be addressed most simply by considering cellular structures that are linear, so that their size can be defined by a single parameter: length . We compare existing studies on several linear biological structures including prokaryotic flagella and flagellar hooks, eukaryotic flagella, sarcomere thin filaments, and microvilli . In some cases, existing evidence strongly supports the idea that length control involves a molecular ruler, in which the size of the overall structure is compared with the size of an individual molecule . In other cases, length control is likely to involve a steady-state balance of assembly and disassembly, in which one or the other rate is inherently length dependent . The lessons learned from size control in linear structures should be applicable to organelles with more complex three-dimensional structures . Expected online publication date for the Annual Review of Cell and Developmental Biology Volume 20 is October 6, 2004 . Please see for revised estimates. Annu Rev Cell Dev Biol . 2004 Jun 11; {Epub ahead of print} New Developments in Mitochondrial Assembly; Koehler CM; The mitochondrion has developed an elaborate translocation system for the import of nuclear-coded proteins and the export of proteins coded on the mitochondrial genome . Precursor proteins contain targeting and sorting information to reach the mitochondrion, while the translocons recognize the information and direct the precursor to the correct compartment . The outer membrane contains the TOM (translocase of the outer membrane) complex for translocation and the SAM (sorting and assembly machinery) complex for assembly of outer membrane proteins with complex topologies . At the inner membrane, the TIM23 (translocase of the inner membrane) mediates the import of mitochondrial proteins with a typical N-terminal targeting sequence, and the TIM22 complex mediates the import of polytopic inner membrane proteins . Based on its prokaryotic origin, the inner membrane also contains several components that mediate the export and assembly of proteins from within the matrix . Together the translocation and assembly complexes coordinate assembly of the mitochondrion . Expected online publication date for the Annual Review of Cell and Developmental Biology Volume 20 is October 6, 2004 . Please see for revised estimates. Annu Rev Genet . 2004 Aug 13; {Epub ahead of print} Comparative Genomic Structure of Prokaryotes; Bentley SD et al.; Recent advances in DNA-sequencing technologies have made available an enormous resource of data for the study of bacterial genomes . The broad sample of complete genomes currently available allows us to look at variation in the gross features and characteristics of genomes while the detail of the sequences reveal some of the mechanisms by which these genomes evolve . This review aims to describe bacterial genome structures according to current knowledge and proposed hypotheses . We also describe examples where mechanisms of genome evolution have acted in the adaptation of bacterial species to particular niches . Expected online publication date for the Annual Review of Genetics Volume 38 is November 10, 2004 . Please see for revised estimates. Annu Rev Microbiol . 2004 Mar 26; {Epub ahead of print} Selection for Gene Clustering by Tandem Duplication; Reams AB et al.; In prokaryotic genomes, related genes are frequently clustered in operons and higher-order arrangements that reflect functional context . Organization emerges despite rearrangements that constantly shuffle gene and operon order . Evidence is presented that the tandem duplication of related genes acts as a driving evolutionary force in the origin and maintenance of clusters . Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments . Clustered genes confer selective benefits via their ability to be coamplified . During evolution, rearrangements that bring together related genes can be selected if they increase the fitness of the organism in which they reside . Similarly, the benefits of gene amplification can prevent the dispersal of existing clusters . Examples of frequent and spontaneous amplification of large genomic fragments are provided . The possibility is raised that tandem gene duplication works in concert with horizontal gene transfer as interrelated evolutionary forces for gene clustering . Expected online publication date for the Annual Review of Microbiology Volume 58 is September 8, 2004 . Please see for revised estimates. Nucleic Acids Res, 2004 Sep 07, 32(16), 4725 - 31 Print 2004. Gene co-regulation is highly conserved in the evolution of eukaryotes and prokaryotes; Snel B et al.; Differences between species have been suggested to largely reside in the network of connections among the genes . Nevertheless, the rate at which these connections evolve has not been properly quantified . Here, we measure the extent to which co-regulation between pairs of genes is conserved over large phylogenetic distances; between two eukaryotes Caenorhabditis elegans and Saccharomyces cerevisiae, and between two prokaryotes Escherichia coli and Bacillus subtilis . We first construct a reliable set of co-regulated genes by combining various functional genomics data from yeast, and subsequently determine conservation of co-regulation in worm from the distribution of co-expression values . For B.subtilis and E.coli, we use known operons and regulons . We find that between 76 and 80% of the co-regulatory connections are conserved between orthologous pairs of genes, which is very high compared with previous estimates and expectations regarding network evolution . We show that in the case of gene duplication after speciation, one of the two inparalogous genes tends to retain its original co-regulatory relationship, while the other loses this link and is presumably free for differentiation or sub-functionalization . The high level of co-regulation conservation implies that reliably predicted functional relationships from functional genomics data in one species can be transferred with high accuracy to another species when that species also harbours the associated genes. Front Biosci, 2004 Sep 01, 9, 3262 - 7 A report on single exon genes (SEG) in eukaryotes; Sakharkar MK et al.; Single exon genes (SEG) are archetypical of prokaryotes . Hence, their presence in intron-rich, multi-cellular eukaryotic genomes is perplexing . Consequently, a study on SEG origin and evolution is important . Towards this goal, we took the first initiative of identifying and counting SEG in nine completely sequenced eukaryotic organisms--four of which are unicellular (E . cuniculi, S . cerevisiae, S . pombe, P . falciparum) and five of which are multi-cellular (C . elegans, A . thaliana, D . melanogaster, M . musculus, H . sapiens) . This exercise enabled us to compare their proportion in unicellular and multi-cellular genomes . The comparison suggests that the SEG fraction decreases with gene count (r = -0.80) and increases with gene density (r = 0.88) in these genomes . We also examined the distribution patterns of their protein lengths in different genomes. Front Biosci, 2004 Sep 01, 9, 2964 - 71 Can ends justify the means? Digging deep for human fusion genes of prokaryotic origin; Yiting Y et al.; Gene fusion has been described as an important evolutionary phenomenon . This report focuses on identifying, analyzing, and tabulating human fusion proteins of prokaryotic origin . These fusion proteins are found to mimic operons, simulate protein-protein interfaces in prokaryotes, exhibiting multiple functions and alternative splicing in humans . The accredited biological functions for each of these proteins is made available as a database at http://sege.ntu.edu.sg/wester/fusion/ J Eukaryot Microbiol, 2004 Jul-Aug, 51(4), 394 - 401 The microtubule analog protein, FtsZ, in the endosymbiont of trypanosomatid protozoa; Motta MC et al.; Blastocrithidia culicis and Crithidia deanei are trypanosomatids that harbor an endosymbiotic bacterium in their cytoplasm . In prokaryotes, numerous proteins are essential for cell division, such as FtsZ, which is encoded by filament-forming temperature-sensitive (fts) genes . FtsZ is the prokaryotic homolog of eukaryotic tubulin and is present in bacteria and archaea, and has also been identified in mitochondria and chloroplasts . FtsZ plays a key role in the initiation of cytokinesis . It self-assembles into the Z ring, which establishes the division plane during septation . In this study, immunoblotting analysis using a FtsZ polyclonal antibody, revealed a 40-kDa band characteristic of FtsZ in endosymbiont fractions and in whole trypanosomatid homogenates, but not in whole cell extracts of aposymbiotic strains . Confocal microscopy and ultrastructural analysis revealed a specific and dispersed labeling over the endosymbiont . Bars and ring-like structures, which are suggestive of the presence of Z-rings, were never observed, even during the division of the symbiont . This peculiar distribution of FtsZ may represent an arrangement of cytoskeleton protein intermediate between prokaryotic and eukaryotic cells . The endosymbiont ftsz gene was completely sequenced after amplification of DNA from symbiont-bearing trypanosomatids or from pure endosymbiont fractions, using PCR and specific primers . The sequences obtained from the endosymbionts from C . deanei and B . culicis were very similar, and were most closely related to bacteria from the genus Pseudomonas. Planta . 2004 Sep 4; {Epub ahead of print} Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs; Romano A et al.; Medium-chain-length poly-3-( R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters . The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes {B . Rehm et al . (1998) J . Biol Chem 273:24044-24051} as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase . In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato ( Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment . Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column . The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines . We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants. Curr Opin Pharmacol, 2004 Oct, 4(5), 479 - 86 Structural understanding of efflux-mediated drug resistance: potential routes to efflux inhibition; McKeegan KS et al.; The active efflux of cytotoxic drugs mediated by multidrug transporters is the basis of multidrug resistance in prokaryotic and eukaryotic cells . Individual multidrug transporters can be extremely versatile, often exhibiting a staggering range of substrate specificity that can negate the effects of clinically relevant therapies . The effective treatment of bacterial, fungal and protozoan infections, along with certain cancer treatments, has been compromised by the presence of multidrug transporters . Traditionally, advances in the understanding of multidrug transporters have been made through biochemical analyses; more recently, however, fundamental advances have been made with the elucidation of several three dimensional structures of representative multidrug pumps . Biochemical and structural analysis of multidrug pumps could lead to the development of novel 'anti-efflux' therapies. J Virol Methods, 2004 Oct, 121(1), 73 - 8 Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections; Rosati S et al.; Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia . The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life . Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period . Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses . In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections . Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA . The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test. Biochemistry, 2004 Sep 14, 43(36), 11576 - 88 Photochromic biliproteins from the cyanobacterium Anabaena sp . PCC 7120: lyase activities, chromophore exchange, and photochromism in phytochrome AphA; Zhao KH et al.; Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore . The cyanobacterium Anabaena sp . PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA) . (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore . (b) AphA chromophorylation is autocatalytic such as in other phytochromes . (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC) . The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor . (d) Through deletion mutations of aphA, a short sequence IQPHGV {amino acids (aa) 26-31} was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259 . (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes . (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (<or=12 nm) of both the Pr and Pfr forms of truncated chromoproteins. Plant Physiol, 2004 Sep, 136(1), 2532 - 47 Epub 2004 Sep 03. Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development; Sze H et al.; A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H(+) exchanger (CHX) genes in plants using Arabidopsis as a model . The predicted protein (85-95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles . The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4 . Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K(+)/H(+) (KHA1), Na(+)/H(+)-K(+) (GerN) antiport, and ligand-gated ion channel (KefC) . The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven beta-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays . Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues . Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis . An exciting idea is that CHX proteins allow osmotic adjustment and K(+) homeostasis as mature pollen desiccates and then rehydrates at germination . The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals . Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp . japonica, suggesting diversification of CHX in Arabidopsis . These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth. Bioinformatics . 2004 Sep 3; {Epub ahead of print} Genome properties: a system for the investigation of prokaryotic genetic content for microbiology, genome annotation and comparative genomics; Haft DH et al.; MOTIVATION: The presence or absence of metabolic pathways, and structures provides context that makes protein annotation far more reliable . Compiling such information across microbial genomes improves the functional classification of proteins and provides a valuable resource for comparative genomics . RESULTS: We have created Genome Properties to present key aspects of prokaryotic biology using standardized computational methods and controlled vocabularies . Properties reflect gene content, phenotype, phylogeny, and computational analyses . The results of searches using Hidden Markov Models (HMMs) allow many properties to be deduced automatically, especially for families of proteins (equivalogs) conserved in function since their last common ancestor . Additional properties are derived from curation, published reports, and other forms of evidence . Genome Properties has been applied to 156 complete prokaryotic genomes, and is easily mined to find differences between species, correlations between metabolic features and families of uncharacterized proteins, or relationships among properties . AVAILABILITY: Genome Properties can be found at http://www.tigr.org/Genome_Properties. Acta Biochim Biophys Sin (Shanghai), 2004 Sep, 36(9), 589 - 96 Recombinant bivalent vaccine against foot-and-mouth disease virus serotype O/A infection in guinea pig; Yi JZ et al.; In this study, two DNA fragments encoding amino acid (141-160)-(21-140)-(141-160) of the VP1 of FMDV (foot-and-mouth disease virus) serotype O and (138-160)-(21-40)-(138-160) of the serotype A FMDV were chemically synthesized . These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A . The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovine-IgG heavy chain coding sequence . Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses . FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 mg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively . 70% and 57% of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively. Biophys J, 2004 Sep, 87(3), 1686 - 96 Exterior site occupancy infers chloride-induced proton gating in a prokaryotic homolog of the ClC chloride channel; Bostick DL et al.; The ClC family of anion channels mediates the efficient, selective permeation of Cl(-) across the biological membranes of living cells under the driving force of an electrochemical gradient . In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl(-) anion . We infer details of this gating mechanism by studying the free energetics of Cl(-) occupancy in the pore of a prokaryotic ClC homolog . These free energetics were gleaned from 30 ns of molecular dynamics simulation on an approximately 133,000-atom system consisting of a hydrated membrane embedded StClC transporter . The binding sites for Cl(-) in the transporter were determined for the cases where the putative gating residue, Glu(148), was protonated and unprotonated . When the glutamate gate is protonated, Cl(-) favorably occupies an exterior site, S(ext), to form a queue of anions in the pore . However, when the glutamate gate is unprotonated, Cl(-) cannot occupy this site nor, consequently, pass through the pore . An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu(148) . Although this suggests that, for the prokaryotic homolog, protonation of Glu(148) may be the first step in transporting Cl(-) at the expense of H(+) transport in the opposite direction, an evolutionary argument might suggest that Cl(-) opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation . During an additional 20 ns free dynamics simulation, the newly discovered outermost site, S(out), and the innermost site, S(int), were seen to allow spontaneous exchange of Cl(-) ions with the bulk electrolyte while under depolarization conditions. Antioxid Redox Signal, 2004 Oct, 6(5), 825 - 34 Bacterial heme oxygenases; Frankenberg-Dinkel N; The importance of heme oxygenases in heme catabolism, iron utilization, and cellular signaling has been recognized for many years and is a well studied process in eukaryotes . Through the accessibility of an increasing number of bacterial genomes, it has become evident that heme oxygenases are also widespread in prokaryotes . In these organisms, the heme oxygenase reaction serves a similar function as in eukaryotes . A major role of bacterial heme oxygenases has been attributed to acquisition of iron in prokaryotic pathogens, but other functions, such as involvement in phytobilin biosynthesis, have been described . This review summarizes the current state of the art on bacterial heme oxygenase research . The various biological roles of this enzyme in prokaryotes and their biochemical properties will be discussed. Genome Biol . 2004;5(9):R64 . Epub 2004 Aug 26. Comprehensive analysis of pseudogenes in prokaryotes: widespread gene decay and failure of putative horizontally transferred genes; Liu Y et al.; BACKGROUND: Pseudogenes often manifest themselves as disabled copies of known genes . In prokaryotes, it was generally believed (with a few well-known exceptions) that they were rare . RESULTS: We have carried out a comprehensive analysis of the occurrence of pseudogenes in a diverse selection of 64 prokaryote genomes . Overall, we find a total of around 7,000 candidate pseudogenes . Moreover, in all the genomes surveyed, pseudogenes occur in at least 1 to 5% of all gene-like sequences, with some genomes having considerably higher occurrence . Although many large populations of pseudogenes arise from large, diverse protein families (for example, the ABC transporters), notable numbers of pseudogenes are associated with specific families that do not occur that widely . These include the cytochrome P450 and PPE families (PF00067 and PF00823) and others that have a direct role in DNA transposition . CONCLUSIONS: We find suggestive evidence that a large fraction of prokaryote pseudogenes arose from failed horizontal transfer events . In particular, we find that pseudogenes are more than twice as likely as genes to have anomalous codon usage associated with horizontal transfer . Moreover, we found a significant difference in the number of horizontally transferred pseudogenes in pathogenic and non-pathogenic strains of Escherichia coli. Environ Microbiol, 2004 Oct, 6(10), 1042 - 8 Intracellular manganese granules formed by a subsurface bacterium; Glasauer S et al.; The demonstrated ability of prokaryotes to form internal metal oxide particles during active metabolism has been restricted to Fe . Mineral-bound Mn(IV) is a known electron acceptor during dissimilatory metal reduction by Shewanella putrefaciens, yet no internal deposits of Mn have been reported to form during anaerobic respiration . We observed distinct nanometre-sized Mn-rich granules in the cytoplasm when either birnessite or pyrolusite (beta-MnO(2)) served as the electron acceptor during growth . During rapid Mn reduction, additional precipitates of Mn were also observed in the periplasm together with the cytoplasmic granules . The bacteria did not accumulate detectable Mn in the outer membrane during formation of the internal precipitates . This is the first report of an intracellular Mn solid produced by bacteria and coupled anaerobically to DR. Shi Yan Sheng Wu Xue Bao, 2002 Jun, 35(2), 77 - 81 {Expression and characterization of carboxylesterase A2 in E . coli}; Huang J et al.; The most commonly observed change that has been linked to resistance development is the increase in activity of carboxylesterases . The putative mechanism involves an overproduction of this enzyme for the sequestration and the hydroxylation of various organophosphate and carbamate insecticides . Carboxylesterases A2 cDNA was amplified from Culex quinquefasciatus by RT-PCR and sequenced consequently . Target gene was inserted into pET-28a to create prokaryotic expression plasmid pET-EstA2 . When pET-EstA2 was transformed into E . coli BL21, the recombinant was induced by IPTG . A pure recombinant protein was obtained by affinity purification . Compared with carboxylesterase A2 purified from Culex quinquefasciatus, carboxylesterase A2, purified from the product of the transgenic of E . coli, has the same Km, but the Vm was higher than that of it, which shows that carboxylesterase A2, purified from the product of E . coli by affinity, is purer than that from Culex quinquefasciatus . The study on the expression and characterization of carboxylesterase A2 in E . coli is more useful for its future application. J Bacteriol, 2004 Sep, 186(18), 6325 - 6 Recombinant cyclophilins lack nuclease activity; Manteca A et al.; Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis . Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins. Genetics, 2004 Aug, 167(4), 1585 - 95 The high-mobility group A-type protein CarD of the bacterium Myxococcus xanthus as a transcription factor for several distinct vegetative genes; Galbis-Martinez M et al.; CarD is the only reported prokaryotic protein showing structural and functional features typical of eukaryotic high-mobility group A transcription factors . In prokaryotes, proteins similar to CarD appear to be confined primarily to myxobacteria . In Myxococcus xanthus, CarD has been previously shown to act as a positive element in two different regulatory networks: one for light-induced synthesis of carotenoids and the other for starvation-induced fruiting body formation . We have now tested the effect of a loss-of-function mutation in the carD gene (carD1) on the expression of a random collection of lacZ-tagged genes, which are normally expressed in the dark during vegetative growth in rich medium . Our results indicate that CarD plays a significant role in the transcriptional regulation of various indicated genes . The carD1 mutation downregulates some genes and upregulates others . Also reported here is the isolation of several mutations that suppress the strong effect of carD1 on the expression of a particular vegetative gene . One of them (sud-2) also suppresses the effect of carD1 on other vegetative genes and on fruiting-body formation . Thus, CarD and the sud-2 gene product appear to participate in a single mechanism, which underlies various apparently diverse regulatory phenomena ascribed to CarD . Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2004 Jun, 18(2), 122 - 6 {Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient}; Mao HX et al.; BACKGROUND: To construct the full-length complementary DNA of HCV genome from an HCV infected patient . METHODS: Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA . The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions . The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis . And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system . RESULTS: The cDNA covered the near full-length of HCV genome from the patient's serum . The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA . The prokaryotic expressed viral proteins could be identified by patient serum . In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media . CONCLUSION: These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone. Photochem Photobiol, 2004 Jul-Aug, 80, 31 - 5 Expressed sequence tag analysis of the dinoflagellate Lingulodinium polyedrum during dark phase; Tanikawa N et al.; To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light-dark cycle . A total of 4324 5'-end sequence tags were isolated . The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database . Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae . We also isolated three bioluminescence-related (luciferase and two luciferin-binding proteins {LBP}) and 37 photosynthesis-related genes . Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene . These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes. J Gen Physiol, 2004 Sep, 124(3), 203 - 10 A cyclic nucleotide modulated prokaryotic K+ channel; Nimigean CM et al.; A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus . The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified . Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer . As followed by a radioactive (86)Rb(+) flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K(+) channel . Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations . Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening. Trends Biochem Sci, 2004 Sep, 29(9), 469 - 77 Ancestral lipid biosynthesis and early membrane evolution; Pereto J et al.; Archaea possess unique membrane phospholipids that generally comprise isoprenoid ethers built on sn-glycerol-1-phosphate (G1P) . By contrast, bacterial and eukaryal membrane phospholipids are fatty acid esters linked to sn-glycerol-3-phosphate (G3P) . The two key dehydrogenase enzymes that produce G1P and G3P, G1PDH and G3PDH, respectively, are not homologous . Various models propose that these enzymes originated during the speciation of the two prokaryotic domains, and the nature (and even the very existence) of lipid membranes in the last universal common ancestor (cenancestor) is subject to debate . G1PDH and G3PDH belong to two separate superfamilies that are universally distributed, suggesting that members of both superfamilies existed in the cenancestor . Furthermore, archaea possess homologues to known bacterial genes involved in fatty acid metabolism and synthesize fatty acid phospholipids . The cenancestor seems likely to have been endowed with membrane lipids whose synthesis was enzymatic but probably non-stereospecific. Curr Opin Plant Biol, 2004 Oct, 7(5), 499 - 505 Plant response regulators implicated in signal transduction and circadian rhythm; Mizuno T; The so-called 'response regulators' were originally discovered as common components of the widespread histidine (His)-->aspartate (Asp) phosphorelay signal transduction system in prokaryotes . Through the course of evolution, higher plants have also come to employ such prokaryotic response regulators (RRs) for their own signal transduction, such as the elicitation of plant hormone (e.g . cytokinin) responses . Furthermore, plants have evolved their own atypical variants of response regulators, pseudo response regulators (PRRs), which are used to modulate sophisticated biological processes, including circadian rhythms and other light-signal responses . Recent studies using the model plant Arabidopsis thaliana have begun to shed light on the interesting functions of these plant response regulators. Expert Opin Biol Ther, 2004 Sep, 4(9), 1533 - 40 CpG DNA: immunomodulation and remodelling of the asthmatic airway; Jain VV et al.; Asthma is a disorder of increasing severity and prevalence . Present understanding of the pathogenesis of asthma emphasises its inflammatory nature . Unbridled inflammation appears to induce irreversible changes, collectively known as airway remodelling . CpG oligonucleotides are a class of compounds that have been developed from studies of prokaryotic DNA . Bacterial DNA, for example, contains sequence motifs based on the dinucleotides cytosine-guanine (CpG); these motifs are suppressed in mammalian DNA and induce (as part of the innate immune system) inflammation, characterised by the induction of T helper type 1 and regulatory responses . The pattern of inflammation promoted by CpG DNA tends to suppress the cytokine and cellular responses characteristic of asthma and atopic disorders . This has led to the investigation and development of CpG DNA as a novel therapeutic approach for the treatment and prevention of these disorders . In addition to suppressing acute and persistent airway inflammation, CpG DNA also reduces the development of subepithelial collagen deposition, goblet cell hyperplasia/metaplasia, and other aspects of airway remodelling . In this paper, the effects of CpG DNA on asthma inflammation and remodelling are reviewed. Plant Physiol, 2004 Sep, 136(1), 2587 - 608 Epub 2004 Aug 27. AraPerox . A database of putative Arabidopsis proteins from plant peroxisomes; Reumann S et al.; To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S {2004} Plant Physiol 135: 783-800) . About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively . To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags . The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases . In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases . Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid . Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases . The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes. BMC Mol Biol . 2004 Aug 26;5(1):14. Sequence periodicity of Escherichia coli is concentrated in intergenic regions; Hosid S et al.; BACKGROUND: Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property . This genomic feature was demonstrated for many prokaryotic genomes . The Escherichia coli sequences display the period close to 11 base pairs . RESULTS: Here we demonstrate that practically only ApA/TpT dinucleotides contribute to overall dinucleotide periodicity in Escherichia coli . The noncoding sequences reveal this periodicity much more prominently compared to protein-coding sequences . The sequence periodicity of ApC/GpT, ApT and GpC dinucleotides along the Escherichia coli K-12 is found to be located as well mainly within the intergenic regions . CONCLUSIONS: The observed concentration of the dinucleotide sequence periodicity in the intergenic regions of E . coli suggests that the periodicity is a typical property of prokaryotic intergenic regions . We suppose that this preferential distribution of dinucleotide periodicity serves many biological functions; first of all, the regulation of transcription. Proc Natl Acad Sci U S A, 2004 Sep 7, 101(36), 13186 - 91 Epub 2004 Aug 25. A trypanothione-dependent glyoxalase I with a prokaryotic ancestry in Leishmania major; Vickers TJ et al.; Glyoxalase I forms part of the glyoxalase pathway that detoxifies reactive aldehydes such as methylglyoxal, using the spontaneously formed glutathione hemithioacetal as substrate . All known eukaryotic enzymes contain zinc as their metal cofactor, whereas the Escherichia coli glyoxalase I contains nickel . Database mining and sequence analysis identified putative glyoxalase I genes in the eukaryotic human parasites Leishmania major, Leishmania infantum, and Trypanosoma cruzi, with highest similarity to the cyanobacterial enzymes . Characterization of recombinant L . major glyoxalase I showed it to be unique among the eukaryotic enzymes in sharing the dependence of the E . coli enzyme on nickel . The parasite enzyme showed little activity with glutathione hemithioacetal substrates but was 200-fold more active with hemithioacetals formed from the unique trypanosomatid th