DNA Cell Biol, 2002 Mar, 21(3), 163 - 88 Interaction in vitro of type III intermediate filament proteins with triplex DNA; Li G et al.; As previously shown, type III intermediate filaments (IFs) select from a mixture of linear mouse genomic DNA fragments mobile and repetitive, recombinogenic sequences that have also been identified in SDS-stable crosslinkage products of vimentin and DNA isolated from intact fibroblasts . Because these sequences also included homopurine.homopyrimidine (Pu.Py) tracts known to adopt triple-helical conformation under superhelical tension, and because IF proteins are single-stranded (ss) and supercoiled DNA-binding proteins, it was of interest whether they have a particular affinity for triplex DNA . To substantiate this, IF-selected DNA fragments harboring a (Pu.Py) segment and synthetic d(GA)(n) microsatellites were inserted into a vector plasmid and the constructs analyzed for their capacity to interact with IF proteins . Band shift assays revealed a substantially higher affinity of the IF proteins for the insert-containing plasmids than for the empty vector, with an activity decreasing in the order of vimentin > glial fibrillary acidic protein > desmin . In addition, footprint analyses performed with S1 nuclease, KMnO(4), and OsO(4)/bipyridine showed that the (Pu.Py) inserts had adopted triplex conformation under the superhelical strain of the plasmids, and that the IF proteins protected the triple-helical insert sequences from nucleolytic cleavage and chemical modification . All these activities were largely reduced in extent when analyzed on linearized plasmid DNAs . Because intramolecular triplexes (H-DNA) expose single-stranded loops, and the prokaryotic ssDNA-binding proteins g5p and g32p also protected at least the Pu-strand of the (Pu.Py) inserts from nucleolytic degradation, it seemed likely that the IF proteins take advantage of their ssDNA-binding activity in interacting with H-DNA . However, in contrast to g5p and E . coli SSB, they produced no clear band shifts with single-stranded d(GA)(20) and d(TC)(20), so that the interactions rather appear to occur via the duplex-triplex and triplex-loop junctions of H-DNA . On the other hand, the IF proteins, and also g32p, promoted the formation of intermolecular triplexes from the duplex d{A(GA)(20).(TC)(20)T} and d(GA)(20) and d(TC)(20) single strands, with preference of the Py (Pu.Py) triplex motif, substantiating an affinity of the proteins for the triplex structure as such . This triplex-stabilizing effect of IF proteins also applies to the H-DNA of (Pu.Py) insert-containing plasmids, as demonstrated by the preservation of intramolecular triplex-vimentin complexes upon linearization of their constituent supercoiled DNAs, in contrast to poor complex formation from free, linearized plasmid DNA and vimentin . Considering that (Pu.Py) sequences are found near MAR/replication origins, in upstream enhancer and promoter regions of genes, and in recombination hot spots, these results might point to roles of IF proteins in DNA replication, transcription, recombination, and repair. J Comput Biol, 2002, 9(2), 225 - 42 Finding motifs using random projections; Buhler J et al.; The DNA motif discovery problem abstracts the task of discovering short, conserved sites in genomic DNA . Pevzner and Sze recently described a precise combinatorial formulation of motif discovery that motivates the following algorithmic challenge: find twenty planted occurrences of a motif of length fifteen in roughly twelve kilobases of genomic sequence, where each occurrence of the motif differs from its consensus in four randomly chosen positions . Such "subtle" motifs, though statistically highly significant, expose a weakness in existing motif-finding algorithms, which typically fail to discover them . Pevzner and Sze introduced new algorithms to solve their (15,4)-motif challenge, but these methods do not scale efficiently to more difficult problems in the same family, such as the (14,4)-, (16,5)-, and (18,6)-motif problems . We introduce a novel motif-discovery algorithm, PROJECTION, designed to enhance the performance of existing motif finders using random projections of the input's substrings . Experiments on synthetic data demonstrate that PROJECTION remedies the weakness observed in existing algorithms, typically solving the difficult (14,4)-, (16,5)-, and (18,6)-motif problems . Our algorithm is robust to nonuniform background sequence distributions and scales to larger amounts of sequence than that specified in the original challenge . A probabilistic estimate suggests that related motif-finding problems that PROJECTION fails to solve are in all likelihood inherently intractable . We also test the performance of our algorithm on realistic biological examples, including transcription factor binding sites in eukaryotes and ribosome binding sites in prokaryotes. Biotechniques, 2002 May, 32(5), 1178, 1180, 1182 - 7 BiZyme: a novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance; Hansen SG et al.; Recombinant vaccinia virus is a useful and powerful tool for the expression and study of foreign genes . Methods that are currently available for the selection of vaccinia virus recombinants include the restoration of viral plaque-forming phenotype, the replication of viral DNA in the presence of BUdR or mycophenolic acid, and the maturation and propagation of virus under antibiotic selection . Though effective, each of these methods requires several weeks of concerted effort to isolate, purify, and amplify a potential recombinant virus . Here we report the development of a bifunctional enzyme (BiZyme) to simplify and expedite the isolation and purification of vaccinia virus recombinants . This novel selection marker is composed of an in-frame fusion between the genes encoding gfp and the neomycin phosphotransferase enzyme (neo) . Remarkably, expression of the chimeric gfp-neo cassette in the presence of G418 confers both viability and fluorescence to transfected or recombinant virus-infected cells, indicating that both activities are retained within the fusion protein . Therfore, BiZyme was incorporated into a recombination plasmid (pGNR) to enable the concomitant insertion of a foreign gene of interest . Here we demonstrate that this selection/amplification process requires a minimum of 11 days to produce the desired vaccinia virus recombinants . Furthermore, recombinants produced in this fashion have been shown to express both biologically active enzymes and antigenically authenticforeign antigens . In addition to its use in the vaccinia virus vector system, the BiZyme bifunctional selection scheme should be applicable to other eukaryotic and prokaryotic expression systems, simply by coupling it to the appropriate host-specific transcription regulatory signals. Biotechniques, 2002 May, 32(5), 1020, 1022, 1024 - 5 passim Cyclic nucleotide-dependent screening of a lamda expression library for nucleic acid binding activities; Loflin P et al.; Here we describe a modified ligand screening strategy for the expression cloning of mammalian proteins that require the activation of protein kinase cascades to activate ligand binding activity . The manipulation of prokaryotic signaling pathways by the application of appropriate inhibitors or agonists to the nitrocellulose filter during functional screening of standard bacteriophage cDNA expression libraries can permit detection of activities that would not otherwise be found in their active state . We have applied this strategy to a A expression library to clone a novel renal cDNA that exhibits cAMP-dependent RNA binding activity when subsequently tested by ectopic expression in mammalian cells. Proteins, 2002 Jul 1, 48(1), 134 - 40 Simple sequences are rare in the Protein Data Bank; Huntley MA et al.; A simple sequence is abundant in the proteins that have been sequenced to date . But unusual protein features, such as a simple sequence, are not present in the same high frequency within structural databases . A subset of these simple sequences, a group with a highly repetitive nature has been shown to be abundant in eukaryotes but not in prokaryotes . In this study, an examination of the eukaryotic proteins in the Protein Data Bank (PDB) has revealed a large deficiency of low complexity, highly repetitive protein repeats . Through simulated databases of similar samples of eukaryotic proteins taken from the National Center for Biotechnology Information (NCBI) database, it is shown that the PDB contains a significantly less highly repetitive, simple sequence than artificial databases of similar composition randomly derived from NCBI . When the structural data for those few PDB sequences that did contain a highly repetitive simple sequence is examined in detail, it is found that in most cases the tertiary structure is unknown for the regions consisting of a simple sequence . This lack of a simple sequence both in the PDB database and in the structural information suggests that this type of simple sequence may produce disordered structures that make structural characterization difficult . Proteins, 2002 Jul 1, 48(1), 75 - 84 Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity; Kinch LN et al.; Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots . PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation . Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold . Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages . The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups . Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function . Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them . Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information . For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions . Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction . In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world . Biochim Biophys Acta, 2002 May 20, 1597(1), 107 - 14 Conformational change of the N-domain on formation of the complex between the GTPase domains of Thermus aquaticus Ffh and FtsY; Shepotinovskaya IV et al.; The structural basis for the GTP-dependent co-translational targeting complex between the signal recognition particle (SRP) and its receptor is unknown . The complex has been shown to have unusual kinetics of formation, and association in vivo is likely to be dependent on catalysis by the SRP RNA . We have determined conditions for RNA-independent association of the 'NG' GTPase domains of the prokaryotic homologs of the SRP components, Ffh and FtsY, from Thermus aquaticus . Consistent with previous studies of the Escherichia coli proteins, the kinetics of association and dissociation are slow . The T . aquaticus FtsY is sensitive to an endogenous proteolytic activity that cleaves at two sites--the first in a lengthy linker peptide that spans the interface between the N and G domains, and the second near the N-terminus of the N domain of FtsY . Remarkably, this second cleavage occurs only on formation of the Ffh/FtsY complex . The change in protease sensitivity of this region, which is relatively unstructured in the FtsY but not in the Ffh NG domain, implies that it undergoes conformational change on formation of the complex between the two proteins . The N domain, therefore, participates in the interactions that mediate the GTP-dependent formation of the targeting complex. FEMS Microbiol Lett, 2002 Apr 9, 209(2), 255 - 60 Recurrent intragenomic recombination leading to sequence homogenization during the evolution of the lipoyl-binding domain; Omelchenko MV et al.; The lipoyl-binding domain is often present, in one or several copies, in the E2 subunit and, less often, in the E1 and E3 subunits of 2-oxo acid dehydrogenase complexes . Phylogenetic analysis shows evidence of multiple, independent intragenomic recombination events between different versions of the lipoyl-binding domain in various bacteria and eukaryotic mitochondria, leading to homogenization of the sequences of the lipoyl-binding domain within the same enzymatic complex in several bacterial lineages . This appears to be the first case of sequence homogenization at the level of an individual domain in prokaryotes. FEMS Microbiol Rev, 2002 Mar, 26(1), 49 - 71 Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence; Sleator RD et al.; Two general strategies exist for the growth and survival of prokaryotes in environments of elevated osmolarity . The 'salt in cytoplasm' approach, which requires extensive structural modifications, is restricted mainly to members of the Halobacteriaceae . All other species have convergently evolved to cope with environments of elevated osmolarity by the accumulation of a restricted range of low molecular mass molecules, termed compatible solutes owing to their compatibility with cellular processes at high internal concentrations . Herein we review the molecular mechanisms governing the accumulation of these compounds, both in Gram-positive and Gram-negative bacteria, focusing specifically on the regulation of their transport/synthesis systems and the ability of these systems to sense and respond to changes in the osmolarity of the extracellular environment . Finally, we examine the current knowledge on the role of these osmostress responsive systems in contributing to the virulence potential of a number of pathogenic bacteria. Int J Biochem Cell Biol, 2002 Aug, 34(8), 983 - 91 Effects of removal of the N-terminal amino acid residues on the activity and conformation of firefly luciferase; Wang XC et al.; A series of deletion mutants were constructed using polymerase chain reaction (PCR) to investigate the roles of luciferase N-terminal residues . The coding sequences of the first 0 (Luc0), 6 (Luc6), 7 (Luc7), 8 (Luc8), 9 (Luc9), 10 (Luc10) and 20 (Luc20) amino acids of the N-terminus were deleted and inserted into the prokaryotic expression vector pBV220 . The results showed that the enzymes were completely inactivated when the first eight or more N-terminal amino acids were removed . The recombinant Luc0 and mutants Luc6 and Luc7 were purified to homogeneity by ammonium sulfate precipitation and liquid chromatography for determination of their activity and conformational changes . The activity assay showed that removal of the first six amino acids resulted in 29% loss of enzymatic activity while removal of the first seven amino acids resulted in nearly complete inactivation (with remaining activity <0.5% of the original activity) . Circular dichroism spectra showed no significant secondary structure changes . But the fluorescence emission maximum red-shift indicated some conformational changes . Luc6 and Luc7 were more sensitive to guanidine unfolding than Luc0 . The present result indicated the significant role of Ile7 to the luciferase stability. Biochim Biophys Acta, 2002 Apr 29, 1596(2), 193 - 200 Novel anti-inflammatory compounds from Rubus sieboldii, triterpenoids, are inhibitors of mammalian DNA polymerases; Murakami C et al.; Two anti-inflammatory triterpenoids, tormentic acid (TA) and euscaphic acid (EA), were found from the plant Rubus sieboldii . These triterpenoids showed an inhibitory effect against enzymes involved in replication, such as calf DNA polymerase alpha (pol alpha) and rat DNA polymerase beta (pol beta) . The IC50 values of TA and EA were 37 and 61 microM for pol alpha and 46 and 108 microM for pol beta, respectively . However, TA and EA did not influence the activities of plant DNA polymerases, DNA primase, human immunodeficiency virus type-1 reverse transcriptase, terminal deoxynucleotidyl transferase, any of the prokaryotic DNA polymerases or DNA and RNA metabolic enzymes tested . TA and EA could prevent the growth of BALL-1 cancer cells, and the LD50 values were 11 and 48 microM, respectively . The cells were halted at G1 phase in the cell cycle . The mode of action of the triterpenoids against anti-inflammatory activity and their relationships to the DNA polymerase inhibitory activity and cell growth inhibition were discussed. Chembiochem, 2002 May 3, 3(5), 384 - 97 Body shaping under water stress: osmosensing and osmoregulation of solute transport in bacteria; Morbach S et al.; Fluctuation of external osmolarity is one of the most common types of environmental stress factors for all kind of cells, both of prokaryotic and of eukaryotic origin . Cells try to keep their volume and/or turgor pressure constant; consequently, both a decrease (hypoosmotic stress) and an increase (hyperosmotic stress) of the solute concentration (correctly: increase or decrease in water activity) in the surrounding area, respectively, are challenges for cellular metabolism and survival . A common example from the prokaryotic world is the fate of a soil bacterium that, after a sunny day has dried out the soil (hyperosmotic stress), is suddenly exposed to a drop of distilled water from a rain cloud (hypoosmotic stress) . The immediate and inevitable passive response to the sudden osmotic shift in the surroundings is fast water efflux out of the cell in the former situation and water influx in the latter . In the worst case, these responses may lead to either loss of cell turgor and plasmolysis or to cell burst . In order to overcome such drastic consequences cells have developed effective mechanisms, namely osmoadaptation, to cope with the two different types of osmotic stress . For a graded reaction to osmotic shifts, cells must be able (1) to sense stimuli related to osmotic stress, (2) to transduce corresponding signals to those systems that properly respond (3) by activating transport or enzymatic functions or (4) by changing gene expression profiles . In this review, membrane proteins involved in the cell's active response to osmotic stress are described . Molecular details of structure, function, and regulation of mechanosensitive efflux channels from various organisms, as well as of osmoregulated uptake systems are discussed. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 138 - 42 Mouse restin inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis; Xu R et al.; Restin, a homologous protein of endostatin, was found by Ramchandran et al . It was the C-terminal fragment of type XV collagen . To analysis the inhibition activity of mouse restin on the proliferation of endothelial cells, the cDNA of restin was amplified from the total RNA of the mouse muscle and cloned into the prokaryotic expression plasmid pQE32 . The recombinant protein was expressed in inclusion body with a yield about 60%--70% of total protein . After refolding, the purified recombinant protein specifically inhibits bovine aortic endothelial (BAE) cell proliferation stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner, but the activity of restin was weaker than that of endostatin . Treatment of BAE cell with recombinant restin caused G(1) arrest and apoptosis in BAE cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Mar, 34(2), 143 - 8 Cloning, expression and immunization of the new antigen gene Sj-Ts4 of Schistosoma japonicum; Zhou DM et al.; In order to explore the molecular mechanism of high immune protection against schistosomes infection in animals infected with Trichinella spiralis, and to provide several cross-protective antigen genes for developing anti-schistosomiasis vaccine, a Schistosoma japonicum adult worm cDNA library was immunoscreened using sera taken from mice infected with Trichinella spiralis . Nine positive clones were obtained after 3 rounds of immunoscreening . Among them, Sj-Ts4 represents a novel gene of S . japonicum, which coding for a novel protein of 210 amino acids . The protein has a deduced molecular mass of 23 kD and isoelectric point of 7.72 . Sj-Ts4 was expressed as a glutathione-S-transferase (GST) fusion protein by cloning into the prokaryotic expression vector pGEX-5X-3 . The recombinant Sj-Ts4 protein (rSj-Ts4) was purified and could be recognized by sera of mice infected with S.japonicum . Vaccination of several groups of mice with rSj-Ts4 or rSj-Ts4 incorporated into Freund's complete adjuvant induced high titers of specific IgG antibodies . Two vaccination groups all obtained significant reduction in worm burden (31.36%, 36.80%, P<0.01), compared with the control groups. Science, 2002 May 10, 296(5570), 1127 - 9 Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment; Kaeberlein T et al.; The majority (>99%) of microorganisms from the environment resist cultivation in the laboratory . Ribosomal RNA analysis suggests that uncultivated organisms are found in nearly every prokaryotic group, and several divisions have no known cultivable representatives . We designed a diffusion chamber that allowed the growth of previously uncultivated microorganisms in a simulated natural environment . Colonies of representative marine organisms were isolated in pure culture . These isolates did not grow on artificial media alone but formed colonies in the presence of other microorganisms . This observation may help explain the nature of microbial uncultivability. Science, 2002 May 10, 296(5570), 1064 - 6 Prokaryotic diversity--magnitude, dynamics, and controlling factors; Torsvik V et al.; There are probably millions of species in the microorganismal domains Bacteria and Archaea (the prokaryotes), and we are only just beginning to work out the basic principles governing their distribution and abundance in natural environments . One characteristic that has become clear is that prokaryote diversity in aquatic environments is orders of magnitude less than in sediments and soils . Hypotheses and models explaining such differences are under development and are beginning to offer promising insights into the mechanisms governing prokaryote diversity and ecosystem function. J Biol Chem, 2002 Jul 26, 277(30), 26779 - 87 Epub 2002 May 09. IMP, GTP, and 6-phosphoryl-IMP complexes of recombinant mouse muscle adenylosuccinate synthetase; Iancu CV et al.; Prokaryotes have a single form of adenylosuccinate synthetase that controls the committed step of AMP biosynthesis, but vertebrates have two isozymes of the synthetase . The basic isozyme, which predominates in muscle, participates in the purine nucleotide cycle, has an active site conformation different from that of the Escherichia coli enzyme, and exhibits significant differences in ligand recognition . Crystalline complexes presented here of the recombinant basic isozyme from mouse show the following . GTP alone binds to the active site without inducing a conformational change . IMP in combination with an acetate anion induces major conformational changes and organizes the active site for catalysis . IMP, in the absence of GTP, binds to the GTP pocket of the synthetase . The combination of GTP and IMP results in the formation of a stable complex of 6-phosphoryl-IMP and GDP in the presence or absence of hadacidin . The response of the basic isozyme to GTP alone differs from that of synthetases from plants, and yet the conformation of the mouse basic and E . coli synthetases in their complexes with GDP, 6-phosphoryl-IMP, and hadacidin are nearly identical . Hence, reported differences in ligand recognition among synthetases probably arise from conformational variations observed in partially ligated enzymes. J Biol Chem, 2002 Jul 26, 277(30), 26886 - 92 Epub 2002 May 09. Reconstitution of a disulfide isomerization system; Collet JF et al.; Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides . In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins . To be active as isomerases, DsbC and DsbG must be kept reduced . This task is performed by the cytoplasmic membrane protein DsbD . DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains . The beta domain is membrane-embedded, whereas the alpha and gamma domains are localized to the periplasm . It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains . To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains . Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG . We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG . We also determined the redox potential of the gamma domain to be -241 mV, and that of the alpha domain was found to be -229 mV . This shows that the direction of electron flow within DsbD is thermodynamically driven. RNA, 2002 Mar, 8(3), 370 - 81 A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA; Xing F et al.; Dihydrouridine modification of tRNA is widely observed in prokaryotes and eukaryotes, as well as in some archaea . In Saccharomyces cerevisiae every sequenced tRNA has at least one such modification, and all but one have two or more . We have used a biochemical genomics approach to identify the gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w), using yeast pre-tRNA(Phe) as a substrate . Dus1 is a member of a widespread family of conserved proteins, three other members of which are found in yeast: YNR015w, YLR405w, and YLR401c . We show that one of these proteins, Dus2, encoded by ORF YNR015w, has activity with two other substrates: yeast pre-tRNA(Tyr) and pre-tRNA(Leu) . Both Dus1 and Dus2 are active as a single subunit protein expressed and purified from Escherichia coli, and the activity of both is stimulated in the presence of flavin adenine dinucleotide . Dus1 modifies yeast pre-tRNA(Phe) in vitro at U17, one of the two positions that are known to bear this modification in vivo . Yeast extract from a dus1-A strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-delta and dus2-delta strains is significantly depleted in dihydrouridine content. Crit Rev Microbiol, 2002, 28(1), 61 - 77 Control of the bacterial cell cycle by cytoplasmic growth; Koch AL; For free-living single-celled organisms, it can be assumed that it is their success in acquiring resources and converting them into cytoplasm that controls the timing of their cell cycles . Cytoplasm is the sink for the bulk of the environmental resources . It must be the case that this type of control must operate in dilute cultures under adequate nutrition in a constant environment . It follows that there ought to be mechanisms that measure or count the cell's biomass or some component of the cytoplasm to measure their growth success . Besides sensing their biomass, they need to know when a certain value of the cell size has been achieved . When this critical state has been achieved, the cell needs to have an all-or-none trigger that either initiates chromosome replication, the completion of cell replication, cell division, or the process of separating sister cells physiologically or physically . Any of these four different stages, in principle, may be the one triggered in response to cell growth in different species of microorganisms . Alternatively, multiple triggers at different cell sizes may be activated at different cell cycle stages . Although initiation of chromosome replication has been believed to be the event triggered in Escherichia coli, this probably is not generally the case and other control mechanisms may act in other prokaryotes . How the increase in cell biomass is self-assessed and used to carry out critical cell cycle events is not understood in any case . This deficiency in our knowledge of microbial cell physiology is grave . The factor that probably has prevented the elucidation of the mechanisms in any organism is that enzymatic processes deal with concentrations, and a cell cycle trigger must respond to the total amount of material present in a cell . This article discusses the theoretically possible classes of mechanisms for the cell to respond when it has achieved its appropriate critical size . These breakdown into three groups: those mechanisms that assess the total amount of biomass or some special subcellular component, and those that measure the ratio of one component to another component where their two syntheses are differently controlled by cell physiology and morphology, and a third group with some specialized mechanisms. Proteins, 2002 Jun 1, 47(4), 481 - 8 M13 endopeptidases: New conserved motifs correlated with structure, and simultaneous phylogenetic occurrence of PHEX and the bony fish; Bianchetti L et al.; M13 endopeptidase alignments have focused mainly on mammalian sequences and on the active site region defining the catalytic sequence signatures . Aligning all available M13 from bacteria to human on a full-length basis, we have performed a sequence analysis . This enabled us to highlight the origin and function of the M13 PHEX subtype family endopeptidase (phosphate regulating gene with homologies to endopeptidases on the X chromosome) . New evolutionary conserved regions in both prokaryotes and eukaryotes have been detected and eukaryotic-specific regions clearly delineated . Using the recently solved neprilysin structure, we have observed that all new motifs, except one, localize in the spatial vicinity of the previously reported catalytic signatures . Interestingly, a highly hydrophobic pocket containing three newly reported motifs is centered by the C-terminal tryptophan residue . Extensive M13 searches in complete and in progress higher eukaryotic genomes have lead to the identification of Danio rerio as the simplest organism having PHEX . Finally, the human PHEX substrate, the parathyroid hormone-related peptide, PTHrP(107-139), is absent in bony fish: this suggests the existence of further PHEX substrates common to both bony fishes and higher vertebrates . Nature . 2002 May 9;417(6885):137. Microbiology: eukaryotic diversity in Spain's River of Fire; Amaral Zettler LA et al.; The Rio Tinto, known by the Phoenicians as 'Ur-yero', or 'River of Fire', because of its deep red colour and high acidity, flows through the world's largest pyritic belt in southwestern Spain . Surprisingly, eukaryotic microbes are the principal contributors of biomass in this hostile river, which has a pH of 2 and contains much higher concentrations of heavy metals than are typically found in fresh waters . Here we show that the Rio Tinto shows an unexpected degree of eukaryotic diversity and includes new lineages that we have identified by sequence analysis of genes encoding small-subunit ribosomal RNAs . The diversity of these eukaryotes is much greater than that of prokaryotes, whose metabolism is responsible for the extreme environment. Nucleic Acids Res, 2002 May 15, 30(10), 2212 - 23 Connected gene neighborhoods in prokaryotic genomes; Rogozin IB et al.; A computational method was developed for delineating connected gene neighborhoods in bacterial and archaeal genomes . These gene neighborhoods are not typically present, in their entirety, in any single genome, but are held together by overlapping, partially conserved gene arrays . The procedure was applied to comparing the orders of orthologous genes, which were extracted from the database of Clusters of Orthologous Groups of proteins (COGs), in 31 prokaryotic genomes and resulted in the identification of 188 clusters of gene arrays, which included 1001 of 2890 COGs . These clusters were projected onto actual genomes to produce extended neighborhoods including additional genes, which are adjacent to the genes from the clusters and are transcribed in the same direction, which resulted in a total of 2387 COGs being included in the neighborhoods . Most of the neighborhoods consist predominantly of genes united by a coherent functional theme, but also include a minority of genes without an obvious functional connection to the main theme . We hypothesize that although some of the latter genes might have unsuspected roles, others are maintained within gene arrays because of the advantage of expression at a level that is typical of the given neighborhood . We designate this phenomenon 'genomic hitchhiking' . The largest neighborhood includes 79 genes (COGs) and consists of overlapping, rearranged ribosomal protein superoperons; apparent genome hitchhiking is particularly typical of this neighborhood and other neighborhoods that consist of genes coding for translation machinery components . Several neighborhoods involve previously undetected connections between genes, allowing new functional predictions . Gene neighborhoods appear to evolve via complex rearrangement, with different combinations of genes from a neighborhood fixed in different lineages. Biol Cell, 2002 Feb, 94(1), 29 - 35 New magnet-sensitive structures in bacterial and archaeal cells; Vainshtein M et al.; The objects of the investigation were: distribution of intracellular magnet-sensitive structures among different taxonomic groups of prokaryotes, localisation and organisation of the magnet-sensitive inclusions (MsI) in cells . The MsI were discovered in representatives of both prokaryotic domains (Bacteria and Archaea), 2 kingdoms and 7 orders of bacteria . They were some amorphous or non-crystalline globules with the electron-transparent centre surrounded with an electron-dense homogenous matrix . The magnetic nature of the structures was shown by attraction with an applied magnet both for the cell suspensions and for the MsI isolated and separated from the destroyed cells . The MsI were studied with transparent electron microscopy and with X-ray analyses . When the cells were grown in the iron-containing nutrient medium, the matrix was enriched with iron . It was shown also that some bacteria grown with cobalt or with chromium contained the cobalt- or chromium-enriched magnetic inclusions. Plant Mol Biol, 2002 Mar-Apr, 48(5-6), 805 - 20 Phylogenetic analysis of the acetyl-CoA carboxylase and 3-phosphoglycerate kinase loci in wheat and other grasses; Huang S et al.; We have applied a two-gene system based on the sequences of nuclear genes encoding multi-domain plastid acetyl-CoA carboxylase (ACCase) and plastid 3-phosphoglycerate kinase (PGK) to study grass evolution . Our analysis revealed that these genes are single-copy in most of the grass species studied, allowing the establishment of orthologous relationships between them . These relationships are consistent with the known facts of their evolution: the eukaryotic origin of the plastid ACCase, created by duplication of a gene encoding the cytosolic multi-domain ACCase gene early in grass evolution, and the prokaryotic (endosymbiont) origin of the plastid PGK . The major phylogenetic relationships among grasses deduced from the nucleotide sequence comparisons of ACCase and PGK genes are consistent with each other and with the milestones of grass evolution revealed by other methods . Nucleotide substitution rates were calculated based on multiple pairwise sequence comparisons . On a relative basis, with the divergence of the Pooideae and Panicoideae subfamilies set at 60 million years ago (MYA), events leading to the Triticum/Aegilops complex occurred at the following intervals: divergence of Lolium (Lolium rigidum) at 35 MYA, divergence of Hordeum (Hordeum vulgare) at 11 MYA and divergence of Secale (Secale cereale) at 7 MYA . On the same scale, gene duplication leading to the multi-domain plastid ACCase in grasses occurred at 129 MYA, divergence of grass and dicot plastid PGK genes at 137 MYA, and divergence of grass and dicot cytosolic PGK genes at 155 MYA . The ACCase and PGK genes provide a well-understood two-locus system to study grass phylogeny, evolution and systematics. Mol Cell Biol, 2002 Jun, 22(11), 3707 - 17 tRNAs in Trypanosoma brucei: genomic organization, expression, and mitochondrial import; Tan TH et al.; The mitochondrial genome of Trypanosoma brucei does not encode tRNAs . Consequently, all mitochondrial tRNAs are imported from the cytosol and originate from nucleus-encoded genes . Analysis of all currently available T . brucei sequences revealed that its genome carries 50 tRNA genes representing 40 different isoacceptors . The identified set is expected to be nearly complete since all but four codons are accounted for . The number of tRNA genes in T . brucei is very low for a eukaryote and lower than those of many prokaryotes . Using quantitative Northern analysis we have determined the absolute abundance in the cell and the mitochondrion of a group of 15 tRNAs specific for 12 amino acids . Except for the initiator type tRNA(Met), which is cytosol specific, the cytosolic and the mitochondrial sets of tRNAs were qualitatively identical . However, the extent of mitochondrial localization was variable for the different tRNAs, ranging from 1 to 7.5% per cell . Finally, by using transgenic cell lines in combination with quantitative Northern analysis it was shown that import of tRNA(Leu)(CAA) is independent of its 5'-genomic context, suggesting that the in vivo import substrate corresponds to the mature, fully processed tRNA. J Drug Target, 2002 Feb, 10(1), 47 - 54 Differential behaviour of fluid liposomes toward mammalian epithelial cells and bacteria: restriction of fusion to bacteria; Desjardins A et al.; Previous work demonstrated that fluid liposomes developed in our laboratory are able to fuse with bacterial outer membranes . This fusion improved the penetration and activity of liposome-encapsulated antibiotics and antisense oligonucleotides into the bacterial cells . Because it is anticipated that fluid liposome encapsulated antibiotics will be administered by aerosols to patients with chronic pulmonary infections or cystic fibrosis (CF), we conducted comparative studies in E . coli, P . aeruginosa and human lung epithelial cells using lipid-mixing assays to investigate the possibility that fluid liposomes might fuse with surrounding epithelial cells . After a 2 h incubation at 4 and 37 degrees C, no fusion between fluid liposomes and human lung epithelial cells was observed, whereas mean levels of 71 and 37% of fusion were observed at 37 degrees C with E . coli and P . aeruginosa cells, respectively . No fusion was observed at 4 degrees C in any cells . A kinetic study where temperature was gradually increased from 7 to 37 degrees C indicated that the fusion process in the two bacteria starts between 28 and 31 degrees C with a mean fusion rate of 0.60%/min at 31 degrees C to reach 1.18%/min at 37 degrees C . The present work suggests that it is unlikely that fluid liposomes fuse with host cells lining the human respiratory tract and further elucidates the fusogenic properties of fluid liposomes with respect to prokaryotes and eukaryotes. J Immunol, 2002 May 15, 168(10), 4951 - 9 Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain; Riedl P et al.; Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes . When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity . HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable . Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity . HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells . The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag . Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag . Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency . Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity . Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine . Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity. Biochem J, 2002 Jul 1, 365(Pt 1), 13 - 8 Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase); Aghajanian S et al.; The final two enzymes in the CoA biosynthetic pathway, phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) and dephospho-CoA kinase (DPCK; EC 2.7.1.24), are separate proteins in prokaryotes, but exist as a bifunctional enzyme in pig liver . In the present study we have obtained sequence information from purified pig-liver enzyme, and identified the corresponding cDNA in a number of species . The human gene localizes to chromosome 17q12-21 and contains regions with sequence similarity to the monofunctional Escherichia coli DPCK and PPAT . The recombinant 564-amino-acid human protein confirmed the associated transferase and kinase activities, and gave similar kinetic properties to the wild-type pig enzyme. Front Biosci, 2002 May 01, 7, d1338 - 46 Mycoplasmosis and immunity of fish and reptiles; Brown DR; Advances in molecular phylogenetics have enabled reconstruction of the most likely chronology of events in prokaryotic evolution and correlation with the paleontologic record with increasing precision . Mycoplasmas probably evolved from clostridial ancestors by genome reduction leading to obligate parasitism of host cells . The vertebrate hosts present at the time of the origin of mycoplasmas about 400 million years ago were fish, and later amphibians and reptiles, whose descendants possess most elements of vertebrate innate and adaptive immunity . Successful colonization of those poikilothermous ("cold-blooded") hosts must have involved adaptation to those defenses, shaping mycoplasma-host interactions for more than 125 million years before the earliest emergence of mammals . That history illuminates one aspect of the potential significance of mycoplasmosis of poikilothermous vertebrates to health and disease of other hosts including humans. Mol Biotechnol, 2002 May, 21(1), 19 - 37 Quantitative analysis of gene expression by reverse transcription polymerase chain reaction and capillary electrophoresis with laser-induced fluorescence detection; Richards MP et al.; There has been a dramatic expansion of DNA sequence information compiled over the past several years for a variety of eukaryotic and prokaryotic genomes . Accompanying this increase in knowledge of genomic structure and organization has been a growing interest in studying the function of individual genes including regulation of their expression . A number of methods such as Northern blotting, ribonuclease protection assay, and hybridization arrays have been developed to analyze gene expression at the transcriptional (mRNA) level . Although quantitative estimates of mRNA transcripts can be obtained from each of these methods, oftentimes they lack sufficient sensitivity or the methodology is too costly or too labor-intensive to be applied to the analysis of a large number of samples . The most sensitive method for analyzing gene expression at the mRNA level involves the combination of reverse transcription and polymerase chain reaction (RT-PCR) . However, in order to provide accurate quantitative estimates of gene expression, a rapid and efficient method is required for separation and detection of the double-stranded DNA (dsDNA) products of RT-PCR . Recent advances in capillary electrophoresis with laser-induced fluorescence detection (CE/LIF) have made this method suitable for the automated analysis of large numbers of RT-PCR samples . An overview of the application of CE/LIF to quantitative analysis of gene expression by RT-PCR is presented along with selected protocols and examples . Both relative-quantitative (RQ) and quantitative-competitive (QC) approaches to RT-PCR are discussed in conjunction with the use of CE/LIF for rapid and accurate quantitative analysis of PCR products. Vestn Ross Akad Med Nauk, 2002, (3), 48 - 51 {Fundamental and applied aspects of neuroendocrine regulation of pro- and eukaryote interactions}; Stadnikov AA et al.; The paper presents the results of light, atomic-force, and electron microscopic studies of prokaryotes (various Staphylococcus aureus strains) and eukaryotes (Rat digestive and respiratory cells and tissues) in in vivo and in vivo interactions . It is concluded that hypothalamic nonapeptides play a modulating role in the persistence and symbiotic relations in the bacterium-host system and regulate cellular and tissue homeostasis through reparative histogenesis. Microbiology, 2002 May, 148(Pt 5), 1389 - 96 Evidence of intermolecular recombination between extrachromosomal DNAs in phytoplasma: a trigger for the biological diversity of phytoplasma? Nishigawa H, Oshima K, Kakizawa S, Jung HY, Kuboyama T, Miyata S, Ugaki M, Namba S. Recombination among bacterial extrachromosomal DNAs (EC-DNAs) plays a major evolutionary role by creating genetic diversity, and provides the potential for rapid adaptation to new environmental conditions . Previously, a 7 kbp EC-DNA, EcOYW1, with a geminivirus-like rolling-circle-replication protein (Rep) gene was isolated and characterized from an original wild-type line (OY-W) of onion yellows (OY) phytoplasma, an endocellular cell-wall-less prokaryote that inhabits the cytoplasm of both plant and insect cells . EcOYW1, found in OY-W, was not present in a mild-symptom line (OY-M) derived from OY-W . A 4 kbp EC-DNA, pOYW, was also isolated and characterized from OY-W, and its pLS1-plasmid-like rep gene was expressed . This paper describes the isolation and sequencing of an EC-DNA of 5560 nt, EcOYW2, from OY-W, and its counterpart EC-DNA of 5025 nt, EcOYM, from OY-M . EcOYW2 and EcOYM contained seven and six ORFs, respectively . They both encoded a geminivirus-like Rep and a putative single-stranded-DNA-binding protein (SSB) . Southern blot analysis indicated that no more EC-DNAs with a rep gene exist in either OY-W or OY-M, which means that the complete set of EC-DNAs has been cloned from the OY-W and OY-M lines of OY phytoplasmas . Sequence analysis revealed that both EcOYW2 and EcOYM have chimeric structures of previously characterized EcOYW1 and pOYW, suggesting that they have a recombinational origin . This is the first evidence of intermolecular recombination between EC-DNAs in phytoplasma . The possible implications of these findings in increasing the biological diversity of phytoplasma are discussed. Biochim Biophys Acta, 2002 Mar 19, 1561(1), 47 - 64 Structure and association of ATP-binding cassette transporter nucleotide-binding domains; Kerr ID; ATP-binding cassette transporters are responsible for the uptake and efflux of a multitude of substances across both eukaryotic and prokaryotic membranes . Members of this family of proteins are involved in diverse physiological processes including antigen presentation, drug efflux from cancer cells, bacterial nutrient uptake and cystic fibrosis . In order to understand more completely the role of these multidomain transporters an integrated approach combining structural, pharmacological and biochemical methods is being adopted . Recent structural data have been obtained on the cytoplasmic, nucleotide-binding domains of prokaryotic ABC transporters . This review evaluates both these data and the conflicting implications they have for domain communication in ABC transporters . Areas of biochemical research that attempt to resolve these conflicts will be discussed. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 69 - 73 {Establishment of a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase gene}; Gao C et al.; BACKGROUND: To establish a sandwich ELISA method for detection of reporter chloramphenicol acetyltransferase (CAT) gene . METHODS: The full length sequence of CAT gene was amplified with PCR using plasmid pBLCAT6 as template, and inserted into the prokaryotic expression plasmid Pgex-2T . The purified fusion protein was emulsified with complete or incomplete Freund adjuvant and injected subcutaneously into rabbits . The antibody was labeled with biotin, and a sandwich ELISA technique with biotin streptavidin amplify system was established . Several CAT reporter plasmids containing different HPV 16 LCR sequences were generated and transfected transiently to monolayer cells in vitro . The cytoplasm proteins were extracted and the expressions of CAT were evaluated with the newly established ELISA assay . RESULTS: SDS-PAGE displayed that the molecular weight of the expressed fusion protein was about 54,000 . The prepared antiserum was able to recognize the CAT protein expressed by mammalian cells or prokaryote cells . Under the control of different promoters and their regulate sequences,two to eight folds CAT expression increased were evaluated in transiently transfected mammalian cells by the newly established sandwich ELISA method . CONCLUSIONS: The established method could sensitively reflect the activities of the upstream promoters, as well as the influence of exchanges of nucleotides within the regulate region on the promoter activities . Therefore, it proposes a convenient assay for the studies using CAT as the reporter gene. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Mar, 16(1), 31 - 2 {Preliminary evidence that a hepatitis E virus (HEV) ORF2 recombinant protein protects cynomolgus macaques against challenge with wild-type HEV}; Bi S et al.; BACKGROUND: To observe the protective effect of hepatitis E virus (HEV) ORF2 recombinant protein expressed in prokaryote cell cynomolgus macaques (cynos) against challenging with wild-type HEV . METHODS: Cynos were immunized with HEV ORF2 recombinant protein and then challenged with wild-type HEV, the unimmunized cynos were used as control . Blood samples were collected and tested to see if there were dynamic changes of ALT and antibody to HEV before and after challenge with wild-type HEV . RESULTS: All the five unimmunized cynos re-presented hepatitis 3 weeks after challenging with wild-type HEV . However, all the five immunized cynos showed no hepatitis and pathological changes . CONCLUSIONS: Cynos can be efficiently protected by immunization with HEV ORF2 recombinant protein against wild-type HEV . This protein can be a promising candidate for HEV vaccine. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2001 Dec, 15(4), 305 - 8 {Analysis and applications of a PrP antibody elicited by synthesized human PrP peptides}; Sun X et al.; BACKGROUND: To further study the immuno-reactivity and specificity of a PrP antibody elicited by synthesized human PrP peptides . METHODS: Construction of a plasmid which expresses a N-terminus deleted human PrP protein and purification of this truncated protein . The prepared PrP antibody was tested for reaction with various prokaryotic expressed human PrP proteins,including the full-length protein,the N-terminus-and C-terminus-truncated proteins with Western blot . The antibody was also used in the immunofluorescence assays to identify the PrP proteins expressed from the cell lines transfected with human PrP genes . Moreover,the brain tissues from scrapie agents-infected hamsters and a CJD patient were extracted and the proteinase K-resistant PrP-res were analyzed with the prepared antibody . RESULTS: The PrP-peptide elicited antibody could recognize both prokaryotic and eukaryotic expressed human PrP proteins, as well as the PrP-res proteins in the brain tissues both from the scrapie-infected hamsters and CJD patient . CONCLUSIONS: The tested antibody possesses eligible immuno-reactivity and specificity and can be used for diagnosis of CJD in the clinical work. Protein Eng, 2002 Apr, 15(4), 337 - 45 Engineering a novel secretion signal for cross-host recombinant protein expression; Tan NS et al.; Protein secretion is conferred by a hydrophobic secretion signal usually located at the N-terminal of the polypeptide . We report here, the identification of a novel secretion signal (SS) that is capable of directing the secretion of recombinant proteins from both prokaryotes and eukaryotes . Secretion of fusion reporter proteins was demonstrated in Escherichia coli, Saccharomyces cerevisiae and six different eukaryotic cells . Estrogen-inducibility and secretion of fusion reporter protein was demonstrated in six common eukaryotic cell lines . The rate of protein secretion is rapid and its expression profile closely reflects its intracellular concentration of mRNA . In bacteria and yeast, protein secretion directed by SS is dependent on the growth culture condition and rate of induction . This secretion signal allows a flexible strategy for the production and secretion of recombinant proteins in numerous hosts, and to conveniently and rapidly study protein expression. J Biol Chem, 2002 Jul 26, 277(30), 26879 - 85 Epub 2002 Apr 30. Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide . Catalase-like activity, compound III formation, and enzyme inactivation; Hiner AN et al.; The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied . Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)) . The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h . The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1) . Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity . A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2) . A similar model is applicable to horseradish peroxidase . The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the {compound I.H(2)O(2)} complex . Inactivation does not occur from compound III . All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily. Dev Genes Evol, 2002 Apr, 212(3), 152 - 7 Epub 2002 Feb 28. The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins; Siegmund T et al.; Many prokaryotic and eukaryotic DNA-binding proteins use a helix-turn-helix (HTH) structure for DNA recognition . Here we describe a new family of eukaryotic HTH proteins, the Pipsqueak (Psq) family, which includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates . Three subgroups of the Psq family can be distinguished . Like the HTH proteins of the prokaryotic resolvase family, members of the CENP-B/transposase subgroup catalyze site-specific recombination reactions . This functional conservation, together with a primary sequence similarity between the resolvase and Psq DNA-binding domains, suggests that the resolvase and Psq families are evolutionarily linked . More than half of the newly identified Drosophila Psq proteins contain a BTB protein-protein interaction domain . All proteins of this BTB subgroup belong to the conserved Tramtrack group of BTB-domain proteins . About half of the members of the Tramtrack group contain a Psq domain, while the other half is made up of proteins that contain a zinc finger domain . Thus, nearly all members of this group appear to be DNA-binding proteins . Among other developmental regulators, the Drosophila cell death protein E93 was found to contain a Psq motif and to define a third subgroup of Psq domain proteins . The high sequence conservation of the E93 Psq motif allowed the identification of E93 orthologs in humans and lower metazoans. J Biol Chem, 2002 Jul 12, 277(28), 25692 - 6 Epub 2002 Apr 25. The location of plastocyanin in vascular plant photosystem I; Ruffle SV et al.; We have studied the binding sites of the electron donor and acceptor proteins of vascular plant photosystem I by electron microscopy/crystallography . Previously, we identified the binding site for the electron acceptor (ferredoxin) . In this paper we complete these studies with the characterization of the electron donor (plastocyanin) binding site . After cross-linking, plastocyanin is detected using Fourier difference analysis of two dimensionally ordered arrays of photosystem I located at the periphery of chloroplast grana . Plastocyanin binds in a small cavity on the lumenal surface of photosystem I, close to the center and with a slight bias toward the PsaL subunit of the complex . The recent release of the full coordinates for the cyanobacterial photosystem I reaction center has allowed a detailed comparison between the structures of the eukaryotic and prokaryotic systems . This reveals a very close homology, which is particularly striking for the lumenal side of photosystem I. Environ Microbiol, 2002 Feb, 4(2), 125 - 9 Effect of oil pollution on euendolithic cyanobacteria of the Arabian Gulf; Al-Thukair AA; Microbial euendoliths (true borer) cyanobacteria are carbonate-boring microorganisms found in modern and ancient marine environments . Modern euendoliths include a wide range of prokaryotes as well as eukaryotes, which have been reported world-wide . The importance of euendolithic cyanobacteria concerns their role in bio-erosion of calcium carbonate substrates and as ecological indicators of shallow, tropical and subtropical marine environments . Arabian Gulf ooids from four sites along the east coast of Saudi Arabia have been bored and inhabited by several species of euendolithic cyanobacteria . This assemblage of different species exists simultaneously within the same ooid grain . Comparisons of 1989 and 1992 data reveal a drastic reduction in active euendoliths, and the average numbers of colonies in these ooids . This study reveals the harmful effect of the 1991 oil spill on these unique microorganisms residing in these ooids. J Biol Chem, 2002 Jul 12, 277(28), 25668 - 76 Epub 2002 Apr 22. The conserved helix C region in the superfamily of interferon-gamma /interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK; Vandenbroeck K et al.; HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins . The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78 . DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the alpha-helical homodimeric secretory cytokine interferon-gamma (IFN-gamma) . We screened solid-phase peptide libraries from human and mouse IFN-gamma to identify DnaK-binding sites . Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN-gamma dimer interface . Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays . No DnaK-binding sites were found in the loops connecting the alpha-helices . The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-gamma, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs . These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain . We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface. J Biol Chem, 2002 Jun 28, 277(26), 23882 - 7 Epub 2002 Apr 22. Indolmycin resistance of Streptomyces coelicolor A3(2) by induced expression of one of its two tryptophanyl-tRNA synthetases; Kitabatake M et al.; Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials . Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain . Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes . We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive . The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant . Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo . Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme . Tryptophan recognition (assessed by k(cat)/K(M)) by TrpRS1 is 4-fold lower than that of TrpRS2 . Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin. Exp Cell Res, 2002 May 1, 275(2), 200 - 14 Exogenous expression of heat shock protein 90kDa retards the cell cycle and impairs the heat shock response; Zhao C et al.; The 90-kDa heat shock protein, HSP90, is an abundant molecular chaperone which functions in cellular homeostasis in prokaryotes and eukaryotes . It is well known that HSP90 plays a critical and indispensable role in regulating cell growth through modulations of various signal transduction pathways, but its roles in cell cycle control are not so well known . We transferred human HSP90 (wild-type or mutated types) expression vectors into NIH-3T3 cells in order to study certain functions of HSP90 in the cell cycle and cell growth under physiological conditions . We found that the exogenous expression of HSP90 (wild-type) induced a decrease in cell growth via retardation of the G1/S transition . The inhibition of cell growth was caused by reduced expressions of cyclin D3 and cyclin A mRNA and protein . On the other hand, no stable transfectants with the three types of mutated HSP90 were obtained . Unexpectedly, exogenous HSP90 expression impaired the heat shock response by inhibiting both heat shock transcription factor 1(HSF1) activation and transportation of HSF1 into the nucleus . The HSF1 function was disrupted by the direct association between HSF1 and exogenous HSP90, which was present as a monomer . These results reveal important roles of HSP90 in cell cycle control and in the stress response of nontransformed cells. Mol Biol (Mosk), 2002 Mar-Apr, 36(2), 277 - 85 {New insights into the molecular mechanisms of evolution: stress increases genetic diversity}; Vel'kov VV; The mechanisms of stress-induced mutagenesis in prokaryotes and realization of reserved (preaccumulated) genetic variation in eukaryotes are considered . In prokaryotes, replication becomes error-prone in stress because of the induction of the SOS response and the inactivation of the mismatch repair system; stress also increases the transposition rate and the efficiency of interspecific gene transfer . In eukaryotes, chaperone HSP90, which restores the native folding of mutant proteins (e.g., signal transduction and morphogenetic proteins) in normal conditions, fails to do so in stress, which leads to abrupt expression of multiple mutations earlier reserved in the corresponding genes . The role of these mechanisms in the evolution of prokaryotes and eukaryotes is discussed. J Cell Biochem, 2002, 85(4), 678 - 88 Yeast methionine aminopeptidase type 1 is ribosome-associated and requires its N-terminal zinc finger domain for normal function in vivo; Vetro JA et al.; Methionine aminopeptidase type 1 (MetAP1) cotranslationally removes N-terminal methionine from nascent polypeptides, when the second residue in the primary structure is small and uncharged . Eukaryotic MetAP1 has an N-terminal zinc finger domain not found in prokaryotic MetAPs . We hypothesized that the zinc finger domain mediates the association of MetAP1 with the ribosomes and have reported genetic evidence that it is important for the normal function of MetAP1 in vivo . In this study, the intracellular role of the zinc finger domain in yeast MetAP1 function was examined . Wild-type MetAP1 expressed in a yeast map1 null strain removed 100% of N-terminal methionine from a reporter protein, while zinc finger mutants removed only 31-35% . Ribosome profiles of map1 null expressing wild-type MetAP1 or one of three zinc finger mutants were compared . Wild-type MetAP1 was found to be an 80S translational complex-associated protein that primarily associates with the 60S subunit . Deletion of the zinc finger domain did not significantly alter the ribosome profile distribution of MetAP1 . In contrast, single point mutations in the first or second zinc finger motif disrupted association of MetAP1 with the 60S subunit and the 80S translational complex . Together, these results indicate that the zinc finger domain is essential for the normal processing function of MetAP1 in vivo and suggest that it may be important for the proper functional alignment of MetAP1 on the ribosomes . Protein Sci, 2002 May, 11(5), 1274 - 7 A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases; Dlakic M; The replication fork blocks are common in both prokaryotes and eukaryotes . In most cases, these blocks are associated with increased levels of mitotic recombination . One of the best-characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae . It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S . cerevisiae, but the mechanistic aspects of these events are still poorly understood . Sequence profile searches revealed that Fob1p is related to retroviral integrases . Subsequently, the catalytic domain of HIV-1 integrase was used as a template to build a reliable three-dimensional model of Fob1p . Structural insights from this study may be useful in explaining Fob1p-mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA. J Virol, 2002 May, 76(10), 4836 - 47 Open reading frame UL26 of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain; Stamminger T et al.; A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells . This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene . With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts . One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain . In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain . By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators . Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells . Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts . By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection . Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected . Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle. Mol Microbiol, 2002 Apr, 44(1), 1 - 8 Oxidative protein folding in bacteria; Collet JF et al.; Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously . Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides . Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB . DsbB generates disulphides de novo from oxidized quinones . These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport . Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing . Two isomerases exist in Escherichia coli, DsbC and DsbG . The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form . DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction. Environ Microbiol, 2002 Jan, 4(1), 29 - 41 Deciphering the action of aromatic effectors on the prokaryotic enhancer-binding protein XylR: a structural model of its N-terminal domain; Devos D et al.; The prokaryotic enhancer-binding protein XylR is the central regulator of the toluene degradation pathway in Pseudomonas species . Copious genetic and biochemical data indicate that the N-terminal domain of the protein (domain A) interacts directly with m-xylene, which renders the protein competent as a transcriptional activator . Single-site and shuffling mutants of XylR or homologues have been reported to change or expand their effector profiles . Here, we follow a fold recognition approach to generate three-dimensional models of the domain A of XylR and DmpR with the purpose of deciphering the molecular activity of this protein family . The model is based on the crystallographic data of the rat catechol O-methyltransferase, a typical alpha/beta fold, consisting of eight alpha-helices and seven beta-strands . The fold identification is supported by physico-chemical properties of conserved amino acids, distribution of residues characteristic of the sequence families and confrontation with experimental data . The model not only provides a rationale for understanding published experimental data, but also suggests the molecular mechanism of the activation step and is a potentially useful conceptual tool for designing regulators with predefined inducer specificities. Pharmacogenomics, 2002 Jan, 3(1), 19 - 30 The pharmacogenetics of NAT: structural aspects; Pompeo F et al.; Arylamine N-acetyltransferases (NATs) catalyze the transfer of an acetyl group from acetyl-CoA to arylhydrazines and to arylamine drugs and carcinogens or to their N-hydroxylated metabolites . NAT plays an important role in detoxification and metabolic activation of xenobiotics and was first identified as the enzyme responsible for inactivation of the antitubercular drug isoniazid, an arylhydrazine . The rate of inactivation was polymorphically distributed in the population: the first example of interindividual pharmacogenetic variation . Polymorphism in NAT activity is primarily due to single nucleotide polymorphisms (SNPs) in the coding region of NAT genes . NAT enzymes are widely distributed in eukaryotes and genome sequences have revealed many homologous members of this enzyme family in prokaryotes . The structures of S almonella typhimurium and Mycobacterium smegmatis NATs have been determined, revealing a unique fold in which a catalytic triad (Cys-His-Asp) forms the active site . Determination of prokaryotic and eukaryotic NAT structures could lead to a better understanding of their role in xenobiotics and endogenous metabolism. Cell Death Differ, 2002 Apr, 9(4), 394 - 404 Origin and evolution of eukaryotic apoptosis: the bacterial connection; Koonin EV et al.; The availability of numerous complete genome sequences of prokaryotes and several eukaryotic genome sequences provides for new insights into the origin of unique functional systems of the eukaryotes . Several key enzymes of the apoptotic machinery, including the paracaspase and metacaspase families of the caspase-like protease superfamily, apoptotic ATPases and NACHT family NTPases, and mitochondrial HtrA-like proteases, have diverse homologs in bacteria, but not in archaea . Phylogenetic analysis strongly suggests a mitochondrial origin for metacaspases and the HtrA-like proteases, whereas acquisition from Actinomycetes appears to be the most likely scenario for AP-ATPases . The homologs of apoptotic proteins are particularly abundant and diverse in bacteria that undergo complex development, such as Actinomycetes, Cyanobacteria and alpha-proteobacteria, the latter being progenitors of the mitochondria . In these bacteria, the apoptosis-related domains typically form multidomain proteins, which are known or inferred to participate in signal transduction and regulation of gene expression . Some of these bacterial multidomain proteins contain fusions between apoptosis-related domains, such as AP-ATPase fused with a metacaspase or a TIR domain . Thus, bacterial homologs of eukaryotic apoptotic machinery components might functionally and physically interact with each other as parts of signaling pathways that remain to be investigated . An emerging scenario of the origin of the eukaryotic apoptotic system involves acquisition of several central apoptotic effectors as a consequence of mitochondrial endosymbiosis and probably also as a result of subsequent, additional horizontal gene transfer events, which was followed by recruitment of newly emerging eukaryotic domains as adaptors. Curr Opin Infect Dis, 2000 Feb, 13(1), 37 - 45 Biology of trichomonosis; Lehker MW et al.; Trichomonas vaginalis is emerging as a major pathogen of men and women and is associated with serious health consequences . Advances in diagnosis and treatment are presented . The complexity of trichomonad pathogenesis is illustrated in the interaction of this parasite with human cells, tissues and the immune system . It is now becoming evident that the interaction of trichomonads with the host is frequently modulated by environmental signals . The molecular biology of trichomonads is still in its infancy, but analysis of genes, genomic structure and transcriptional mechanisms suggest that trichomonads combine both prokaryotic and eukaryotic features . Evidence for the ancient divergence of trichomonads from other eukaryotic lineages is discussed. Curr Opin Allergy Clin Immunol, 2001 Dec, 1(6), 549 - 54 Can knowledge of the molecular structure of allergens improve immunotherapy? Pomes A, Chapman MD. Conventional immunotherapy may be associated with the development of adverse reactions, including anaphylaxis, due to the use of increasing doses of allergen . Standardization of extracts is necessary in order to assess the correct amount of allergen administered . In recent years, increased knowledge on the molecular structure of allergens has allowed the development of novel alternatives for immunotherapy . Initially, allergens were cloned and expressed as recombinant proteins in eukaryotic and prokaryotic systems . Crystallization of the purified proteins led to the elucidation of the tertiary structure of the allergen . Molecular biology techniques were used to construct modified allergens whose new IgE binding properties were studied . IgE antibody mapping combined with molecular modeling has allowed the recognition of IgE binding sites on the surface of the molecule . This information has been applied to the engineering of new modified allergens, with and without adjuvants, that retain immunogenicity but with reduced allergenicity . The use of these molecules for immunotherapy should allow the administration of greater doses of allergen, without the undesired side effects characteristic of conventional immunotherapy. Biophys J, 2002 May, 82(5), 2627 - 34 First evidence for phototropin-related blue-light receptors in prokaryotes; Losi A et al.; A prokaryotic protein, YtvA from Bacillus subtilis, was found to possess a light, oxygen, voltage (LOV) domain sharing high homology with the photoactive, flavin mononucleotide (FMN)-binding LOV domains of phototropins (phot), blue-light photoreceptors for phototropism in higher plants . Computer-based three-dimensional modeling suggests that YtvA-LOV binds FMN in a similar pocket as phot-LOVs . Recombinant YtvA indeed exhibits the same spectroscopical features and blue-light-induced photochemistry as phot-LOVs, with the reversible formation of a blue-shifted photoproduct, assigned to an FMN-cysteine thiol adduct (Thio383) . By means of laser-flash photolysis and time-resolved optoacoustic experiments, we measured the quantum yield of formation for Thio383, Phi(Thio) = 0.49, and the enthalpy change, DeltaH(Thio) = 135 kJ/mol, with respect to the parent state . The formation of Thio383 is accompanied by a considerable volume contraction, DeltaV(Thio) = -13.5 ml/mol . Similar to phot-LOVs, Thio383 is formed from the decay of a red-shifted transient species, T650, within 2 micros . In both YtvA and free FMN, this transient has an enthalpy content of approximately 200 kJ/mol, and its formation is accompanied by a small contraction, DeltaV(T) approximately -1.5 ml/mol, supporting the assignment of T650 to the FMN triplet state, as suggested by spectroscopical evidences . These are the first studies indicating that phototropin-related, blue-light receptors may exist also in prokaryotes, besides constituting a steadily growing family in plants. J Gen Virol, 2002 May, 83(Pt 5), 979 - 90 Cloning and sequencing of TT virus genotype 6 and expression of antigenic open reading frame 2 proteins; Kakkola L et al.; The near-full-length genome of a TT virus (TTV) (HEL32), closely related to the previously uncharacterized genotype 6, was cloned and sequenced . The genomic organization of HEL32 was compared to 41 published near-full-length TTV sequences representing 17 genotypes . In the majority of genomes, the open reading frame (ORF) 2 region was divided into two separate ORFs, 2a and 2b . The ORF2a sequence was conserved among all genotypes, while the ORF2b region showed more variability . The two corresponding putative proteins of HEL32 were expressed in prokaryotes and their antigenic potential was studied . IgM and IgG antibodies to the respective ORF2-encoded proteins, fp2a and fp2b, and the presence of TTV DNA were studied in the sera of 89 constitutionally healthy adults . By immunoblot using the small TTV proteins as antigens, strong IgM and IgG reactivities were found in 9 and 10% of subjects, respectively . Follow-up studies for 12-15 years of three subjects showed either a persistent coexistence of IgM and TTV DNA or the appearance of viral DNA regardless of pre-existing antibodies . The low prevalence of IgG could be due to the weak immunogenicity of these probably non-structural proteins or to a genotype-specific antibody response . By nested PCR of the conserved ORF2a region, the prevalence of TTV DNA was 85% . TTV genotype 6 sequences were found by specific PCR in 3 of 35 (8.6%) subjects . The low prevalence of TTV IgG compared to the high TTV DNA prevalence, the coexistence of antibodies and viral DNA and the appearance of TTV DNA regardless of pre-existing antibodies suggest that the B-cell immunity against these minor TTV proteins would not be cross protective. Proc Natl Acad Sci U S A, 2002 Apr 30, 99(9), 6428 - 33 Epub 2002 Apr 16. Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants; Maser P et al.; Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins . These transporters contain four loop domains in one polypeptide with a proposed distant homology to K(+) channel selectivity filters . Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na(+)-coupled K(+) transport . Arabidopsis AtHKT1, however, transports only Na(+) in eukaryotic expression systems . To understand the molecular basis of this difference we constructed a series of AtHKT1/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K(+) selectivity . A single-point mutation, Ser-68 to glycine, was sufficient to restore K(+) permeability to AtHKT1 . The reverse mutation in HKT1, Gly-91 to serine, abrogated K(+) permeability . This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K(+) channel selectivity filter GYG motif . The importance of such filter glycines for K(+) selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2 . Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na(+) transporters in plants and that Na(+)/K(+) symport in HKT proteins is associated with a glycine in the filter residue . These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K(+) channels. FEMS Microbiol Lett, 2002 Feb 5, 207(2), 185 - 92 Cloning and characterization of a major surface protein (MspTL) of Treponema lecithinolyticum associated with rapidly progressive periodontitis; Park KK et al.; The gene encoding a major surface protein (MspTL) of Treponema lecithinolyticum, a periodontopathogen, was cloned and sequenced . The mspTL gene has a 1770-bp open reading frame (ORF) encoding a protein of 590 amino acids with a predicted molecular mass of 65 kDa which had a typical prokaryotic signal sequence (19 amino acids) . MspTL showed a high level of homology with major sheath protein (MspA) of Treponema maltophilum, phylogenetically the closest relative of T . lecithinolyticum . Southern blot analysis indicated that the mspTL gene exists in a single copy and Northern blot analysis showed that the mspTL transcript is monocistronic . Another ORF located downstream of mspTL was in the same orientation and encoded a putative protein, in which the first N-terminal 291 amino acids were identified . The homologous region of this protein is also a part on the T . maltophilum mspA locus. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jan, 34(1), 57 - 61 {Site-directed mutagenesis of the cysteines of human IL-18 and its effect on IL-18 activity}; Pei DS et al.; To study the structure-function relationships of human IL-18(hIL-18), site-directed mutagenesis was used to generate four hIL-18 cysteine mutants, C74S, C104S, C112S and C163S . The cDNAs of the four cysteine mutants were inserted into prokaryotic expression vector pJW2 and expressed as inclusion bodies in E . coli . The inclusion bodies were washed with 2 mol/L urea, dissolved in 8 mol/L urea, and purified by chromatography on Sephadex G-100 column . The purity of the purified mutants were greater than 90% as judged by SDS-PAGE . The activity of rhIL-18 C74S, C104S, C112S and C163S accounted for 5%, 81%, 58% and 11% of wild type, respectively . These results suggest that Cys74 and Cys163 play important roles in inducing IFN-gamma production in human peripheral blood mononuclear cells. Funct Integr Genomics, 2002 Apr, 1(6), 357 - 66 Epub 2001 Dec 11. Two-hybrid-based analysis of protein-protein interactions of the yeast multidrug resistance protein, Pdr5p; Subba Rao G et al.; The ATP-binding cassette (ABC) transporters are a large family of proteins responsible for the translocation of a variety of compounds across the membranes of both prokaryotes and eukaryotes . The inter-protein and intra-protein interactions in these traffic ATPases are still only poorly understood . In the present study we describe, for the first time, an extensive yeast two-hybrid (Y2H)-based analysis of the interactions of the cytoplasmic loops of the yeast pleiotropic drug resistance (Pdr) protein, Pdr5p, an ABC transporter of Saccharomyces cerevisiae . Four of the major cytosolic loops that have been predicted for this protein {including the two nucleotide-binding domain (NBD)-containing loops and the cytosolic C-terminal region} were subjected to an extensive inter-domain interaction study in addition to being used as baits to identify potential interacting proteins within the cell using the Y2H system . Results of these studies have revealed that the first cytosolic loop (CL1)--containing the first NBD domain--and also the C-terminal region of Pdr5p, interact with several candidate proteins . The possibility of an interaction between the CL1 loops of two neighboring Pdr5p molecules was also indicated, which could possibly have implications for dimerization of this protein. Biochim Biophys Acta, 2002 Feb 20, 1574(1), 85 - 92 Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases; Murakami C et al.; Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase . We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon) . Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report . Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively . On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities . RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM . The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I . RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases . As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM . The cells were halted at G1 phase in the cell cycle by RAL. Zhonghua Yi Xue Za Zhi, 2002 Mar, 82(5), 338 - 40 {Anti-apoptosis gene survivin promotes cell growth and transformation}; Zhu H et al.; OBJECTIVE: To investigate the role and molecular mechanism of surviving, an anti-apoptosis gene, in cell growth and transformation . METHODS: Coding sequence of surviving was amplified from Daudi cell mRNA by RT-PCR and then cloned into prokaryotic and eukaryotic vectors . The vectors with surviving were transfected into BL21 cells of Escherichia coli and human embryonic kidney 293 cells . The cells were cultured . Protein was extracted from the cells and examined by gel electrophoresis . Suspension of 293 cells was cultured and the number of cells was counted every other day, thus a growth curve was drawn . Another suspension of 293 cells was cultured in soft agar to observe the number of colonies . Cells transfected with plasmids void of surviving were used as controls . RESULTS: The anti-apoptosis gene surviving was well expressed in BL21 cells and 293 cells . The growth curve showed that the proliferation rate of 293 cells was slightly faster than that of control cells, however, without significant difference . Soft agar assay showed that the colonies formed by surviving-transfected 293 cells were of greater size and with greater number . Western blotting showed overexpression of cyclin D1 and c-myc, two important cancer proteins, in cells transfected with surviving . CONCLUSION: The anti-apoptosis gene surviving promotes cell transformation . These effects may depend on the functions of cyclin D1 and c-myc. Mol Microbiol, 2002 Mar, 43(6), 1565 - 75 Identification of genes that are associated with DNA repeats in prokaryotes; Jansen R et al.; Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses . This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences . To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR) . In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b . The direct repeats and the leader sequences were conserved within a species, but dissimilar between species . The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements . Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes . The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship . The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression . The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes. Plant Physiol, 2002 Apr, 128(4), 1470 - 9 Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii; Theiss C et al.; Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms . In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines . In synchronized C . reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels . Spermine, however, could not be detected in C . reinhardtii cells . Exogenous polyamines caused a decrease in ODC activity . Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase . Most of the cells had already doubled their cell mass after this growth period . The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine . The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period . Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis. Bioessays, 2002 Apr, 24(4), 293 - 6 Bacterial actins? An evolutionary perspective; Doolittle RF et al.; According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life . An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles . Two recent papers1, 2 present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin . Sequence comparisons reveal that eukaryotic actin and the bacterialhomolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories . Biochemistry, 2002 Apr 16, 41(15), 4938 - 45 Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function; Yan W et al.; P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR . Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha) . We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis . P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members . To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain . Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae . This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs . Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70 . However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay . Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system . Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family. Am J Vet Res, 2002 Apr, 63(4), 551 - 8 Effects of equine recombinant interleukin-1alpha and interleukin-1beta on proteoglycan metabolism and prostaglandin E2 synthesis in equine articular cartilage explants; Takafuji VA et al.; OBJECTIVES: To evaluate the effects of equine recombinant interleukin-1alpha (rEqIL-1alpha) and recombinant interleukin-1beta (rEqIL-1beta) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture . SAMPLE POPULATION: Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse . PROCEDURE: Expression constructs containing cDNA sequences encoding EqIL-1alpha and EqIL-1beta were generated, prokaryotically expressed, and the recombinant protein purified . Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL-1alpha or rEqIL-1beta treatments 10 to 500 ng/ml) . Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media . Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan . Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay . Data were collected at 48-hour intervals and normalized by DNA content . RESULTS: Proteoglycan release was induced by rEqIL-1alpha and rEqIL-1beta at concentrations > or =0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively . Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days . Increased PGE2 concentrations were observed at IL-1 concentrations > or = 0.1 ng/ml at 2 and 4 days . CONCLUSIONS AND CLINICAL RELEVANCE: The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro . These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses. Appl Microbiol Biotechnol, 2002 Mar, 58(3), 275 - 85 Epub 2001 Dec 20. Microbial production of vitamin B12; Martens JH et al.; One of the most alluring and fascinating molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti pernicious anemia factor and whose enigmatic complex structure is matched only by the beguiling chemistry that it mediates . The biosynthesis of this essential nutrient is intricate, involved and, remarkably, confined to certain members of the prokaryotic world, seemingly never have to have made the eukaryotic transition . In humans, the vitamin is required in trace amounts (approximately 1 microg/day) to assist the actions of only two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase; yet commercially more than 10 t of B12 are produced each year from a number of bacterial species . The rich scientific history of vitamin B12 research, its biological functions and the pathways employed by bacteria for its de novo synthesis are described . Current strategies for the improvement of vitamin B12 production using modern biotechnological techniques are outlined. Science, 2002 Apr 5, 296(5565), 148 - 51 The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation; Bell SD et al.; The conserved Sir2 family of proteins has protein deacetylase activity that is dependent on NAD (the oxidized form of nicotinamide adenine dinucleotide) . Although histones are one likely target for the enzymatic activity of eukaryotic Sir2 proteins, little is known about the substrates and roles of prokaryotic Sir2 homologs . We reveal that an archaeal Sir2 homolog interacts specifically with the major archaeal chromatin protein, Alba, and that Alba exists in acetylated and nonacetylated forms . Furthermore, we show that Sir2 can deacetylate Alba and mediate transcriptional repression in a reconstituted in vitro transcription system . These data provide a paradigm for how Sir2 family proteins influence transcription and suggest that modulation of chromatin structure by acetylation arose before the divergence of the archaeal and eukaryotic lineages. Bioinformatics, 2002 Mar, 18(3), 470 - 81 STOCKS: STOChastic Kinetic Simulations of biochemical systems with Gillespie algorithm; Kierzek AM; MOTIVATION: The availability of a huge amount of molecular data concerning various biochemical reactions provoked numerous attempts to study the dynamics of cellular processes by means of kinetic models and computer simulations . Biochemical processes frequently involve small numbers of molecules (e.g . a few molecules of a transcriptional regulator binding to one 'molecule' of a DNA regulatory region) . Such reactions are subject to significant stochastic fluctuations . Monte Carlo methods must be employed to study the functional consequences of the fluctuations and simulate processes that cannot be modelled by continuous fluxes of matter . This provides the motivation to develop software dedicated to Monte Carlo simulations of cellular processes with the rigorously proven Gillespie algorithm . RESULTS: STOCKS, software for the stochastic kinetic simulation of biochemical processes is presented . The program uses a rigorously derived Gillespie algorithm that has been shown to be applicable to the study of prokaryotic gene expression . Features dedicated to the study of cellular processes are implemented, such as the possibility to study a process in the range of several cell generations with the application of a simple cell division model . Taking expression of Escherichia coli beta-galactosidase as an example, it is shown that the program is able to simulate systems composed of reactions varying in several orders of magnitude by means of reaction rates and the numbers of molecules involved . AVAILABILITY: The software is available at ftp://ibbrain.ibb.waw.pl/stocksand Supplementary information: Parameters of the model of prokaryotic gene expression are available in example files of software distribution. Curr Opin Microbiol, 2002 Apr, 5(2), 135 - 41 Structure/function relationships in OmpR and other winged-helix transcription factors; Kenney LJ; Response regulators are the output component of two-component regulatory systems, the predominant form of signal transduction systems utilized by prokaryotes . The majority of response regulators function as transcription factors, yet detailed descriptions of their mechanisms of DNA binding and its consequences are lacking . Versatility in the modes of DNA binding is evident with winged helix-turn-helix proteins, raising doubts that mechanisms of DNA binding will be generalizable among members of the family . The current focus of some of the research efforts aimed at understanding activation and DNA binding by response regulators is highlighted in this review. Chembiochem, 2002 Apr 2, 3(4), 274 - 93 Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases; Jeltsch A; DNA methyltransferases catalyze the transfer of a methyl group from S-adenosyl-L-methionine to cytosine or adenine bases in DNA . These enzymes challenge the Watson/Crick dogma in two instances: 1) They attach inheritable information to the DNA that is not encoded in the nucleotide sequence . This so-called epigenetic information has many important biological functions . In prokaryotes, DNA methylation is used to coordinate DNA replication and the cell cycle, to direct postreplicative mismatch repair, and to distinguish self and nonself DNA . In eukaryotes, DNA methylation contributes to the control of gene expression, the protection of the genome against selfish DNA, maintenance of genome integrity, parental imprinting, X-chromosome inactivation in mammals, and regulation of development . 2) The enzymatic mechanism of DNA methyltransferases is unusual, because these enzymes flip their target base out of the DNA helix and, thereby, locally disrupt the B-DNA helix . This review describes the biological functions of DNA methylation in bacteria, fungi, plants, and mammals . In addition, the structures and mechanisms of the DNA methyltransferases, which enable them to specifically recognize their DNA targets and to induce such large conformational changes of the DNA, are discussed. Protein Eng, 2002 Mar, 15(3), 169 - 83 Knowledge-based selection of targets for structural genomics; Frishman D; The problem of rational target selection for protein structure determination in structural genomics projects on microbes is addressed . A flexible computational procedure is described that directly incorporates the whole body of annotation available in the PEDANT genome database into the sequence clustering and selection process in order to identify proteins that are likely to possess currently unknown structural domains . Filtering out gene products based on predicted structural features, such as known three-dimensional structures and transmembrane regions, allows one to reduce the complexity of neighbor relationships between sequences and all but eliminates the need for further partitioning of single-linkage clusters into disjoint protein groups corresponding to homologous families . The results of a large-scale computation experiment in which exemplary target selection for 32 prokaryotic genomes was conducted are presented. Microbiology, 2002 Apr, 148(Pt 4), 1129 - 42 Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases; Johnson CH et al.; Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species . Evidence is presented here for two additional catalase isozymes in H . capsulatum . Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts . Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated . The transcriptional expression of the H . capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA . Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H(2)O(2)) and carbon source (glucose vs glycerol) . Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress . A phylogenetic analysis of presently available catalase sequences and intron residences was done . The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes. J Biol Chem, 2002 Jun 14, 277(24), 21405 - 13 Epub 2002 Apr 03. Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix; Baumann F et al.; The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria . Little is known about the intramitochondrial sorting of these proteins . We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space . Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane . The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane . The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria . A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors. Sheng Li Xue Bao, 2002 Feb 25, 54(1), 23 - 7 {Prokaryotic expression and purification of human hepatic stimulator substance}; Du HJ et al.; To explore the possibility of prokaryotic expression of human hepatic stimulator substance (hHSS), hHSS gene was inserted in the downstream of glutathion S-transferase (GST) in a pET-42a expression vector and recombinant GST-hHSS fusion protein was expressed under IPTG induction in BL-21(DE3) cells . The recombinant HSS was purified with His.Tag affinity chromatography, and its bioactivity was analyzed . The results showed that GST-hHSS fusion protein was expressed both as a soluble or a inclusive body in bacterial cytosol . The soluble GST-hHSS expression reached up to 30% of the whole soluble protein of bacteria as determined by densitometry . The cleavage of GST-hHSS fusion protein with Factor Xa produced two fragments of the protein, which sized 33 and 15 kD, respectively . The molecular weight of recombinant HSS protein was identical to theoretical deduction based on the DNA sequences . The protein homology of 15 kD hHSS could be efficiently eluted out after Factor Xa cleavage . It is further indicated that the recombinant hHSS is able to proliferate hepatoma cells of BEL-7402 in the preliminary experiments. Neuroreport, 2002 Mar 25, 13(4), 437 - 41 HSP90 inhibitors alter capsaicin- and ATP-induced currents in rat dorsal root ganglion neurons; McDowell TS et al.; Heat shock proteins (HSPs) are major components of eukaryotic and prokaryotic cells with particularly high levels of expression in neurons . HSPs control protein folding, transport of proteins to and from the nucleus, incorporation of proteins into the cell membrane, and maintenance of the functional activity of several proteins involved in transcriptional control . In this study we demonstrate that inhibitors of HSP90 alter currents mediated by the ligand gated channels, P2X and VR1 . P2X and VR1 are membrane receptors activated by ATP and capsaicin, respectively, and are thought to be involved in inflammation-related nociception . The HSP90 inhibitors geldanamycin (GLD), radicicol (RAD) herbimycin A (HERB) potentiated ATP induced currents, whereas only GLD altered capsaicin-induced currents in isolated DRG neurons . At low (< 1 microM) concentrations, GLD potentiated the capsaicin-induced current, while at high concentrations (10-25 microM) it inhibited it . The results suggest a potential involvement of HSPs in nociception. Cell Mol Biol (Noisy-le-grand), 2002 Feb, 48(1), 79 - 82 Functional studies of mutations in the human protoporphyrinogen oxidase gene in variegate porphyria; Morgan RR et al.; The autosomal dominant disorder, variegate porphyria (VP), results from mutations in the protoporphyrinogen oxidase (PPOX) gene . We have investigated the effects of 22 disease-associated missense mutations in this gene on enzyme activity . Mutants were generated in the expression plasmid pHPPOX by site-directed mutagenesis . They were screened for PPOX a