Front Biosci, 2000 Sep 01, 5, D768 - 79 Oxygen: friend or foe? Archaeal superoxide dismutases in the protection of intra- and extracellular oxidative stress; Cannio R et al.; Both "environmental chemistry" and metabolic biochemical reactions can constantly generate in vivo free radicals and other oxygen-derived species that can cause severe damage to almost all biomolecules, especially to DNA, proteins, and lipids . The superoxide anion has been shown to be the most readily generated and spread radical among organisms and it is a common intermediate of oxidative stress processes in the cells . The antioxidant defense system of superoxide dismutases (SOD) scavenges and minimizes the formation of this radical, and thus plays a major role in reducing cumulative oxidative damage in different cell compartments both in aerobic and anaerobic cells . In the cell, cytosol SODs are constitutively present and induced by many oxidative agents able to raise the superoxide concentrations . Presence of SODs, however, in extracellular cell-associated locations demonstrates how valuable they are in maintaining the integrity of cells against oxidative stress generated by the cell environment, particularly upon increased oxygenation . Because SODs have recently been found in Archaea, which are prokaryotes, sometimes living in extreme environments, even in anaerobic ones, these enzymes can be considered essential: they may have allowed the evolution of aerobic respiration starting from an ancient form of oxygen-insensitive life. Annu Rev Biochem, 2000, 69, 183 - 215 Two-component signal transduction; Stock AM et al.; Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein . The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein . Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response . The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems . The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained . Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins. Exp Parasitol, 2000 Jul, 95(3), 176 - 86 Trypanosoma cruzi: a putative vacuolar ATP synthase subunit and a CAAX prenyl protease-encoding gene, as examples of gene identification in genome projects; Porcel BM et al.; An international genome program has been initiated to increase the knowledge about the Trypanosoma cruzi genome and thereby find effective tools to treat Chagas' disease . We here report the molecular characterization of two novel genes found in the course of this project . Two of the open reading frames (ORF) identified in the sequencing of the third smallest chromosome of the CL Brener strain of T . cruzi were selected for further molecular characterization due to their similarity to genes with interesting functions in other organisms and their potential as targets to combat the parasite . The first ORF (402 bp) showed homology to a 14-kDa vacuolar ATP synthase subunit F from a variety of organisms, such as yeast, rat, bovine, human, and a number of prokaryotes . The second ORF (1188 bp) resembled a CAAX prenyl protease-encoding gene, identified in different organisms, including Homo sapiens, Saccharomyces cerevisiae, and Arabidopsis thaliana, as well as several prokaryotes . RT-PCR from T . cruzi total epimastigote RNA allowed us to isolate the complete transcripts of these genes . Furthermore, screening of an available normalized cDNA library derived from the same stage of the parasite confirmed that both genes are expressed at least in the epimastigote stage of T . cruzi . Comparison of the putative T . cruzi proteins to their counterparts in other organisms revealed significant protein sequence conservation over large evolutionary distances . Computer analysis revealed the presence of several motifs in both proteins, possibly related to the regulation and localization of these proteins in the parasite . Genomics, 2000 Sep 1, 68(2), 167 - 78 Isolation, characterization and targeted disruption of mouse ppia: cyclophilin A is not essential for mammalian cell viability; Colgan J et al.; Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans . These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the conformation of proteins in cells . CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection . The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA . Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA . Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome . mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere . Sequence analysis of a 369-bp fragment from the proximal promoter region of mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Sp1, AP-2, GATA factors, c-Myb, and NF-IL-6 . This region is sufficient to drive high-level reporter gene expression in transfected cells . Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting . Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mammalian cell viability . J Bacteriol, 2000 Sep, 182(18), 5271 - 3 Bacillus subtilis YvrK is an acid-induced oxalate decarboxylase; Tanner A et al.; Bacillus subtilis has been shown to express a cytosolic oxalate decarboxylase (EC 4.1.1.2) . The enzyme was induced in acidic growth media, particularly at pH 5.0, but not by oxalate . The enzyme was purified, and N-terminal sequencing identified the protein to be encoded by yvrK . The role of the first oxalate decarboxylase to be identified in a prokaryote is discussed. Genome Res, 2000 Aug, 10(8), 1185 - 93 Ecologic genomics of DNA: upstream bending in prokaryotic promoters; Bolshoy A et al.; After our analysis of the distribution of predicted intrinsic curvature along all available complete prokaryotic genomes, the genomes were divided into two groups . Curvature distribution in all prokaryotes of the first group indicated a substantial fraction of promoters characterized by intrinsic DNA curvature located within or upstream of the promoter region . We did not find this peculiar DNA curvature distribution in prokaryotes in the second group . Remarkably, all bacteria of the first group were mesophilic, whereas many prokaryotes of the second group were hyperthermophilic . We hypothesize that DNA curvature plays a biologic role in gene regulation in mesophilic as opposed to hyperthermophilic prokaryotes, i.e., DNA curvature presumably has a functional adaptive significance determined by temperature selection. Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1176 - 9 Cloning, crystallization and preliminary characterization of a beta-carbonic anhydrase from Escherichia coli; Cronk JD et al.; Carbonic anhydrases are zinc metalloenzymes that fall into three distinct evolutionary and structural classes, alpha, beta and gamma . Although alpha-class enzymes, particularly mammalian carbonic anhydrase II, have been the subject of extensive structural studies, for the beta class, consisting of a wide variety of prokaryotic and plant chloroplast carbonic anhydrases, the structural data is quite limited . A member of the beta class from E . coli (CynT2) has been crystallized in native and selenomethionine-labelled forms and multiwavelength anomalous dispersion techniques have been applied in order to determine the positions of anomalous scatterers . The resulting phase information is sufficient to produce an interpretable electron-density map . A crystal structure for CynT2 would contribute significantly to the emerging structural knowledge of a biologically important class of enzymes that perform critical functions in carbon fixation and prokaryotic metabolism. Cell Tissue Res, 2000 Aug, 301(2), 311 - 22 Distribution of brominated compounds within the sponge Aplysina aerophoba: coupling of X-ray microanalysis with cryofixation techniques; Turon X et al.; The major secondary metabolites of the sponge Aplysina aerophoba are brominated compounds . X-ray energy dispersive microanalysis was therefore used to locate secondary metabolites via the Br signal in energy emission spectra from sponge sections . To test the reliability of this method in the face of the loss or redistribution of metabolites during processing, we compared the results obtained by conventional aldehyde fixation with those obtained by cryofixation and cryosubstitution with and without cryoembedding . Bromine appeared to be concentrated in two sponge structures, viz . fibres and spherulous cells, when cryofixed material was examined . However, X-ray microanalysis failed to demonstrate the presence of bromine in spherulous cells in chemically fixed samples, showing the need for cryotechniques to avoid the loss of compounds . Cryofixation plus cryosubstitution methods performed best regarding structural preservation and the immobilization of metabolites . The presence of bromine in the spherulous cells suggests that this cell type is the producer of the secondary metabolites, as described for other sponge species . Nevertheless, the presence of bromine in sponge fibres indicates that they can accumulate metabolic substances, although we have been unable to assess whether the chemicals are in their original form or in a modified state within the fibres . A . aerophoba has both bacterial and cyanobacterial symbionts in its mesohyl; the absence of brominated compounds in them contrasts with previous findings in other sponges with prokaryote symbionts. J Mol Med, 2000, 78(5), 239 - 42 Cytochrome c oxidase assembly factors with a thioredoxin fold are conserved among prokaryotes and eukaryotes; Chinenov YV; Cytochrome c oxidase (COX) is a multi-subunit terminal oxidase of the eukaryotic respiratory chain involved in the reduction of oxygen to water . Numerous lines of evidence suggest that the assembly of COX is a multi-step, assisted process that depends on several assembly factors with largely unknown functions . Sco1/2 proteins have been isolated as high-copy number suppressors of a deletion of copper chaperone Cox 17, implicating Sco1/2 in copper transport to COX subunits I or II . Here I report the similarity of Sco1/2 assembly factors to peroxiredoxins and thiol:disulfide oxidoreductases with a thioredoxin fold, suggesting that Sco-related proteins perform a catalytic rather than a copper transport function . Reported sequence similarities, together with the functional role of bacterial Sco-related proteins suggest that Sco-related proteins represent a new class of membrane-anchored thiol:disulfide oxidoreductases involved in COX maturation. Appl Microbiol Biotechnol, 2000 Jul, 54(1), 84 - 9 Molecular design of novel metal-binding oligomeric human metallothioneins; Hong SH et al.; Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs . An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed . The increased molecular size of the constructs resulted in improved stability and productivity in E . coli . The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apometallothioneins were reconstituted with Cd or Zn ions in a reducing condition . The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245-255 nm . The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein . These novel oligomeric hMTs may be useful in bioremediation for heavy metals. Cell Mol Life Sci, 2000 Jun, 57(6), 896 - 8 Life without myoglobin; Garry DJ et al.; Hemoproteins are widely distributed among prokaryotes, unicellular eukaryotes, plants and animals {1} . Myoglobin, a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers in vertebrates, has been proposed to facilitate oxygen transport to the mitochondria {1-3} . This cytoplasmic hemoprotein was the first protein to be subjected to definitive structural analysis and has been a subject of long-standing and ongoing interest to biologists {1-3} . Recently, we utilized gene disruption technology to generate mice that are viable and fertile despite a complete absence of myoglobin {4} . This unexpected result led us to reexamine existing paradigms regarding the function of myoglobin in striated muscle. J Mol Evol, 2000 Aug, 51(2), 173 - 81 Determining the relative rates of change for prokaryotic and eukaryotic proteins with anciently duplicated paralogs; Kollman JM et al.; The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor . The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase . In each case phylogenetic trees were constructed and the distances determined for all pairs . Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate. J Mol Evol, 2000 Aug, 51(2), 131 - 40 Evolution of simple sequence in proteins; Huntley M et al.; The proteins of Saccharomyces cerevisiae contain a high proportion of low-complexity, simple sequences . These are protein segments composed almost exclusively or largely of a single repetitive amino acid polymer and are the most commonly shared feature between proteins . We have examined a survey of other species to determine how widespread this phenomenon might be . This was done by comparing how frequently segments from one protein are present in other proteins . Any recently evolutionarily related proteins were excluded . It was found that the most commonly shared features of eukaryotic proteins were repetitive but that prokaryotes did not contain such shared, extensively redundant repeats . The proportion of eukaryotic proteins that contain a significantly repetitive fraction changes dramatically from species to species . In addition the individual amino acids present in these repeats change between species . This suggests that the primary sequence of the repeats may not be important for their function . Further tests of the yeast repeats confirmed that these repeats evolve more quickly than the remainder of the protein sequence within which they are embedded . These results show that these rapid evolving, simple sequence repeats are in fact the most commonly shared pattern between all of the genomic proteins of eukaryotes. Infect Immun, 2000 Sep, 68(9), 5062 - 7 Escherichia coli K1 aslA contributes to invasion of brain microvascular endothelial cells in vitro and in vivo; Hoffman JA et al.; Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics . To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary . To identify potential bacterial genes which contribute to E . coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E . coli K1 was mutagenized with TnphoA . TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type . In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia . The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous to E . coli K-12 aslA (arylsulfatase-like gene) . The deduced amino acid sequence of the E . coli K1 aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes . Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion . Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6 . Complementation of the decreased-invasion phenotypes of these mutants was achieved when aslA was supplied in trans . This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E . coli K1. Cancer Immunol Immunother, 2000 Aug, 49(6), 325 - 34 Modulation of interleukin-12 synthesis by DNA lacking the CpG motif and present in a mycobacterial cell wall complex; Filion MC et al.; A mycobacterial cell wall complex prepared from the non-pathogenic microorganism Mycobacterium phlei, where mycobacterial DNA is preserved and complexed to cell wall fragments, possesses anticancer and immunomodulatory activity . DNA from a number of prokaryotes has been found to modulate the immune system and to induce cytokine synthesis . We have therefore determined whether the DNA associated with this complex has the ability to induce the synthesis of interleukin-12 (IL-12), a potent anticancer cytokine . Mycobacterial DNA complexed with cell wall fragments or DNA purified from M . phlei induced IL-12 synthesis by murine and human monocytes and macrophages in vitro, and was capable of inducing IL-12 synthesis in vivo in mice following i.p . administration . Neutralization of DNA with cationic liposomes or digestion with DNase I significantly decreased the ability of the cell wall complex to induce IL-12 . CpG methylation of DNA extracted from these cell walls or from M . phlei did not affect the induction of IL-12 synthesis by monocytes and macrophages . In contrast, CpG methylation of DNA from Escherichia coli abolished its ability to induce IL-12 synthesis . These results demonstrate that unmethylated CpG motifs present in M . phlei DNA are not a prerequisite for the induction of IL-12 synthesis . The size of the mycobacterial DNA, in the range of 5 bp to genomic DNA, did not influence its capacity to induce IL-12 . Our results emphasize that M . phlei DNA associated with the cell wall complex makes a significant contribution to the overall immunomodulatory and anticancer activity of this mycobacterial cell wall preparation and that these activities are not correlated with the presence of CpG motifs. Mutat Res, 2000 Aug 21, 469(1), 51 - 61 Hydrogen peroxide induced mutations at the HPRT locus in primary human T-lymphocytes; Diaz-Llera S et al.; Reactive oxygen species (ROS) produced by intracellular metabolism are believed to contribute to spontaneous mutagenesis in somatic cells . Hydrogen peroxide (H(2)O(2)) has been shown to induce a variety of genetic alterations, probably by the generation of hydroxyl radicals via the Fenton reaction . The kinds of DNA sequence alterations caused by H(2)O(2) in prokaryotic cells have been studied extensively, whereas relatively little is known about the mutational spectrum induced by H(2)O(2) in mammalian genes . We have used the T-cell cloning assay to study the ability of H(2)O(2) to induce mutations at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in primary human lymphocytes . Treatment of cells for 1 h with 0.34-1.35 mM of H(2)O(2) caused a dose dependent decrease of cell survival and increase of the HPRT mutant frequency (MF) . After 8 days of expression time, the highest dose of H(2)O(2) caused a 5-fold increase of MF compared to the untreated control cells . Mutant clones were collected and the genomic rearrangements at the T-cell receptor (TCR) gamma-locus were studied to identify independent mutations . RT-PCR and DNA sequencing was used to identify mutations in the HPRT coding region . Due to a relatively high frequency of sibling clones, only six independent mutations were obtained among the controls, and 20 among the H(2)O(2) treated cells . In both sets, single base pair substitutions were the most common type of mutation (5/6 and 13/20, respectively), with a predominance of transitions at GC base pairs, which is also the most common type of HPRT mutation in T-cells in vivo . Among the single base pair substitutions, five were new mutations not previously reported in the human HPRT mutation database . Overall, the kinds of mutation occurring in T-cells in vivo and H(2)O(2) treated cells were similar, albeit the number of mutants was too small to allow a meaningful statistical comparison . These results demonstrate that H(2)O(2) is mutagenic to primary human T-lymphocytes in vitro and induces mutations of the same kind that is observed in the background spectrum of HPRT mutation in T-cells in vivo. Mutat Res, 2000 Aug 30, 460(3-4), 245 - 56 Structure and function of mismatch repair proteins; Yang W; DNA mismatch repair is required for maintaining genomic stability and is highly conserved from prokaryotes to eukaryotes . Errors made during DNA replication, such as deletions, insertions and mismatched basepairs, are substrates for mismatch repair . Mismatch repair is strand-specific and targets only the newly synthesized daughter strand . To initiate mismatch repair in Escherichia coli, three proteins are essential, MutS, for mismatch recognition, MutH, for introduction of a nick in the target strand, and MutL, for mediating the interactions between MutH and MutS . Homologues of MutS and MutL important for mismatch repair have been found in nearly all organisms . Mutations in MutS and MutL homologues have been linked to increased cancer susceptibility in both mice and humans . Here, we review the crystal structures of the MutH endonuclease, a conserved ATPase fragment of MutL (LN40), and complexes of LN40 with various nucleotides . Based on the crystal structure, the active site of MutH has been identified and an evolutionary relationship between MutH and type II restriction endonucleases established . Recent crystallographic and biochemical studies have revealed that MutL operates as a molecular switch with its interactions with MutH and MutS regulated by ATP binding and hydrolysis . These crystal structures also shed light on the general mechanism of mismatch repair and the roles of Mut proteins in preventing mutagenesis. DNA Cell Biol, 2000 Jul, 19(7), 421 - 9 Comparative study of the coupling between topoisomerase I activity and high-mobility group proteins in E . coli and mammalian cells; Veilleux S et al.; It is now well established that the HMG box DNA-binding motif can alter the topology of double-stranded DNA in several ways . Using the spermatid-specific tsHMG as a model protein of the HMG-1/-2 family, we have demonstrated that its expression in E . coli produces an increase in plasmid supercoiling density that is likely a consequence of its ability to constrain free supercoils in vivo . As demonstrated in vitro, stabilization of free DNA supercoils by tsHMG prevents topoisomerase I from gaining access to the template and could represent a mechanism for the apparent inhibition of topoisomerase I in bacteria . A similar modulation of eukaryotic topoisomerase I activity was not detected after expression of the tsHMG in mammalian cells . This differential response is discussed in terms of the marked difference in DNA packaging and accessibility of free supercoils in prokaryotic vs . eukaryotic cells. Pol Merkuriusz Lek, 2000 May, 8(47), 353 - 5 {Immunogenic Mycobacterium tuberculosis heat shock protein in tuberculosis}; Dubaniewicz A; The heat shock protein (hsp) are produced by prokaryotic and eukaryotic cells in the response to a increase in temperature and a variety of insults . Hsp have been defined by their apparent molecular weight as family: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and SHsp (15-25 kD) . Analysis of the immune response of M . tuberculosis infected individuals allowed for identification of antigens of tubercle bacilli . Most of them belong to evolutionarily highly conserved hsp, which cross-reactive with the hsp of E . Coli . It is conceivable that in a tuberculous granuloma both pathogenes and host cells are stressed and produce hsp . The heat shock proteins with molecular weight of 71 kD, 65 kD, 10-16 kD are immunogens and a functional role as molecular chaperones . The literature on the immunology of hsp in infectious diseases is complex and confusing and it is still not clear whether host immune responses to these proteins are protective, or damaging, as they may lead to the induction of autoimmunity . The study is engaged in a role of others mycobacterial hsp in host immuno-reactivity and their compliance with treatment of infectious diseases, neoplastic genic therapy or/and with more powerful antimycobacterial vaccine. Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1033 - 7 Crystallization and preliminary X-ray analysis of the conserved domain IV of Escherichia coli 4.5S RNA; Jovine L et al.; 4.5S RNA forms with Ffh protein the prokaryotic signal recognition particle (SRP), a highly conserved ribonucleoprotein complex essential for protein secretion . It also independently binds to elongation factor G (EF-G) in the ribosome and has a function in a subset of translocation events that is transient but required for viability . Crystals of three different constructs encompassing the conserved domain IV of 4.5S RNA, containing the recognition elements for both Ffh and EF-G, were obtained . Native X-ray diffraction data were collected for two crystal forms under cryogenic cooling conditions . The best crystals are of a 45 nt construct, diffract anisotropically to 2.6 A resolution using synchrotron radiation and belong to space group P3(2)21, with unit-cell parameters a = b = 69.1, c = 84.6 A and a single RNA molecule per asymmetric unit. Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1030 - 2 Crystallization and preliminary X-ray diffraction studies of the signal recognition particle receptor FtsY from Mycoplasma mycoides; Gariani T et al.; The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system . All four proteins are part of a unique subfamily of GTPases . Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified . Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials . Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E . coli) . A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation . The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit. J Mol Microbiol Biotechnol, 1999 Nov, 1(2), 281 - 7 Modular assembly of voltage-gated channel proteins: a sequence analysis and phylogenetic study; Nelson RD et al.; Voltage-sensitive cation-selective ion channels of the voltage-gated ion channel (VGC) superfamily were examined by a combination of sequence alignment and phylogenetic tree construction procedures . Segments of the alpha-subunits of K+-selective channels homologous to the structurally elucidated KcsA channel of Streptomyces lividans were multiply aligned, and this alignment provided the database for computer-assisted structural analyses and phylogenetic tree construction . Similar analyses were conducted with the four homologous repeats of the alpha-subunits from representative Ca2+- and Na+-selective channels, as well as with the ensemble of K+, Ca2+ and Na+ channels . In both the single subunit of the K+ channels and the individual repeats of the Ca2+ and Na+ channels, the analyses suggest the occurrence of at least two tandemly arranged modules corresponding to the predicted voltage-sensor domain and the pore domain . The phylogenetic analyses reveal strict clustering of segments according to cation-selectivity and repeat unit . We surmise that the pore module of the prokaryotic K+ channel was the primordial polypeptide upon which other modules were superimposed during evolution in order to generate phenotypic diversity . These observations may prove applicable to all members of the VGC family yet to be discovered throughout the prokaryotic and eukaryotic kingdoms. J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 45 - 50 Microbial gene transfer: an ecological perspective; Paul JH; Microbial gene transfer or microbial sex is a means of exchanging loci amongst prokaryotes and certain eukaryotes . Historically viewed as a laboratory artifact, recent evidence from natural populations as well as genome research has indicated that this process may be a major driving force in microbial evolution . Studies with natural populations have taken two approaches- either adding a defined donor with a traceable gene to an indigenous community, and detecting the target gene in the indigenous bacteria, or by adding a model recipient to capture genes being transferred from the ambient microbial flora . However, both approaches usually require some cultivation of the recipient, which may result in a dramatic underestimation of the ambient transfer frequency . Novel methods are just evolving to study in situ gene transfer processes, including the use of green fluorescent protein (GFP)-marked plasmids, which enable detection of transferrants by epifluorescence microscopy . A transduction-like mechanism of transfer from viral-like particles produced by marine bacteria and thermal spring bacteria to Escherichia coil has been documented recently, indicating that broad host range transduction may be occurring in aquatic environments . The sequencing of complete microbial genomes has shown that they are a mosaic of ancestral chromosomal genes interspersed with recently transferred operons that encode peripheral functions . Archaeal genomes indicate that the genes for replication, transcription, and translation are all eukaryotic in complexity, while the genes for intermediary metabolism are purely bacterial . And in eukaryotes, many ancestral eukaryotic genes have been replaced by bacterial genes believed derived from food sources . Collectively these results indicate that microbial sex can result in the dispersal of loci in contemporary microbial populations as well as having shaped the phylogenies of microbes from multiple, very early gene transfer events. J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 23 - 31 Bacterial signals and antagonists: the interaction between bacteria and higher organisms; Rice SA et al.; It is now well established that bacteria communicate through the secretion and uptake of small diffusable molecules . These chemical cues, or signals, are often used by bacteria to coordinate phenotypic expression and this mechanism of regulation presumably provides them with a competitive advantage in their natural environment . Examples of coordinated behaviors of marine bacteria which are regulated by signals include swarming and exoprotease production, which are important for niche colonisation or nutrient acquisition (e.g . protease breakdown of substrate) . While the current focus on bacterial signalling centers on N-Acylated homoserine lactones, the quorum sensing signals of gram-negative bacteria, these are not the only types of signals used by bacteria . Indeed, there appears to be many other types of signals produced by bacteria and it also appears that a bacterium may use multiple classes of signals for phenotypic regulation . Recent work in the area of marine microbial ecology has led to the observation that some marine eukaryotes secrete their own signals which compete with the bacterial signals and thus inhibit the expression of bacterial signalling phenotypes . This type of molecular mimicry has been well characterised for the interaction of marine prokaryotes with the red alga, Delisea pulchra. Vox Sang, 2000, 78 Suppl 2, 57 - 60 DNA vaccines; Reimann J et al.; BACKGROUND AND OBJECTIVES: Humoral and cellular immune responses to protein antigens can be efficiently primed by nucleic acid or DNA vaccination . In DNA-based vaccination, immunogenic proteins are expressed with correct posttranslational modification, conformation or oligomerization; this ensures the integrity of epitopes that stimulate neutralizing antibody (B cell) responses . DNA (or RNA) immunization is exceptionally potent in stimulating T cell responses because antigenic peptides are efficiently generated in (endogenous or exogenous) processing pathways (without interference by viral proteins) from intracellular or extracellular protein antigens expressed after transient in vivo transfection . Both features are difficult to achieve with recombinant subunit vaccines produced in eukaryotic or prokaryotic expression systems . The current state of vector designs, strategies for delivery of DNA vaccines, priming humoral and cellular immune responses by DNA vaccines, experimental strategies facilitated by DNA vaccines, unique advantages of DNA vaccination, experience of DNA vaccination in preclinical animal models and clinical trials, and potential risks of DNA vaccination are discussed . Excellent reviews on DNA-based vaccination have been published recently {1-3}. J Biol Chem, 2000 Oct 20, 275(42), 32585 - 91 A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins; Koc EC et al.; Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis . The proteins in six individual spots were subjected to in-gel tryptic digestion . Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry . The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled . Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified . Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes . These proteins correspond to Escherichia coli S10 and S14 . Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans . A homolog of one of these proteins was observed in D . melanogaster but not in C . elegans, while a homolog of the other was present in C . elegans but not in D . melanogaster . A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome . This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes. J Exp Bot, 2000 Jul, 51(348), 1171 - 8 Primary sodium plasma membrane ATPases in salt-tolerant algae: facts and fictions; Gimmler H; For thermodynamic reasons algae growing in media of both high salinity and high alkalinity require active export of sodium . However, experimental evidence for an active Na+-dependent cycle was scarce until recently, in contrast to the situation in marine bacteria (including cyanobacteria), fungi and animals . However, a review of literature reveals that some progress has been made in this respect, recently: data demonstrate that at least in two marine algae, Tetraselmis (Platymonas) viridis and Heterosigma akashiwo (syn . Olisthodiscus luteus), active Na+-export is carried out by means of a plasma membrane localized Na+-pump (apparent molecular mass 100-140 kDa) . Biochemical characteristics of this vanadate-sensitive, but ouabain-resistant primary P-type Na+-ATPase are described and compared with the corresponding properties of Na+-ATPase from prokaryotes and animals . Alternative mechanisms for Na+-pumping are discussed. Invest Ophthalmol Vis Sci, 2000 Aug, 41(9), 2648 - 57 Heparin's roles in stabilizing, potentiating, and transporting LEDGF into the nucleus; Fatma N et al.; PURPOSE: Lens epithelium-derived growth factor (LEDGF) is a 60-kDa protein that dramatically enhances cellular survival, growth, adhesiveness, and resistance to heat and oxidative stress . Full-size recombinant LEDGF is degraded during prokaryotic preparation . Heparin's capacity to stabilize recombinant LEDGF in the face of various stresses (heat, pH, proteolysis), to potentiate its growth-enhancing properties, and to enable transport of LEDGF into the nucleus of mouse lens epithelial cells has been characterized . METHODS: LEDGF-cDNA was cloned in a pGEX-2T expression vector to produce a fusion protein, GST-LEDGF . Porcine heparin was used to stabilize GST-LEDGF . Heparin-Sepharose was used to characterize heparin-GST-LEDGF binding, and GST-LEDGF or heparin-GST-LEDGF was used to quantitate heparin's stabilization of LEDGF in the face of heat, pH, and proteolytic stresses . Fluorescein isothiocyanate-labeled GST-LEDGF and heparin-GST-LEDGF were incubated with cultured mouse lens epithelial cells (LECs) . Fluorescence microscopy and immunostaining techniques were used to monitor heparin's potentiation of LEDGF's growth stimulation and heparin's role in the translocation of GST-LEDGF from the extracellular space into the cytoplasm and nucleus . RESULTS: Heparin, at concentrations as low as 7.1 mg/ml, protected GST-LEDGF from degradation and increased the yield of the full-size fusion protein in a prokaryotic system . It also protected GST-LEDGF from heat, acid-base deactivation, and proteolytic degradation with trypsin and chymotrypsin and greatly potentiated LEDGF's enhancement of mouse LEC growth in culture . It also increased nuclear uptake of exogenous GST-LEDGF and endogenous LEDGF . CONCLUSIONS: Heparin protected GST-LEDGF from degradation under various stress conditions and facilitated transport of GST-LEDGF into the nucleus. Int J Tissue React, 2000, 22(1), 5 - 13 Biochemical studies of a natural antioxidant isolated from rosemary and its application in cosmetic dermatology; Calabrese V et al.; Oxidative damage to DNA, RNA, proteins and cell membranes occurs when the cellular concentration of reactive oxygen species exceeds the capacity of the cell to eliminate them . Aerobic prokaryotic and eukaryotic organisms have developed a set of cell defense systems to mitigate the damaging effects of reactive oxygen species . Epithelial surfaces contain antioxidants that could be expected to provide a defence against environmental stress caused by reactive oxygen and nitrogen species . Skin, which has a highly differentiated and complex structure, is particularly vulnerable to free radical damage because of its contact with oxygen and with other environmental stimuli . Fruit and vegetables contain several classes of compounds that when ingested can potentially contribute to endogenous modulation of antioxidant defences . The purpose of this study was to investigate the effectiveness of a natural extract derived from rosemary to protect free radical-induced skin damage . We provide evidence that an alcoholic extract of rosemary leaves, Rosm1, is endowed with strong antioxidant activity and, as evaluated by both in vitro and in vivo systems, is capable of inhibiting oxidative alterations to skin surface lipids . The present study provides a preclinical perspective on the interface between the biochemical properties of a natural extract isolated from rosemary leaves, a better understanding of the endogenous antioxidant potential of skin and the real validity of natural antioxidant biotechnology in antiaging skin management. J Mol Graph Model, 2000 Feb, 18(1), 7 - 17, 57-8 Construction of a 3D model of oligopeptidase B, a potential processing enzyme in prokaryotes; Gerczei T et al.; A three dimensional structural model of oligopeptidase B (OpB) was constructed by homology modeling . High resolution X-ray structure of prolyl oligopeptidase (PEP), the only protein with sequential and functional homology was used as a template . Initial models of OpB were built by the MODELLER and were analysed by the PROCHECK programs . The best quality model was chosen for further refinement by two different techniques--either constrained molecular dynamics simulations or simulated annealing calculations starting from 500 K . The overall quality of each of the refined models was evaluated and the simulated annealing procedure found to be more effective . The refined model was analysed by different protein analysis programs including PROCHECK for the evaluation of the Ramachandran plot quality, PROSA for testing interaction energies and WHATIF for the calculation of packing quality . This structure was found to be satisfactory and also stable at room temperature as demonstrated by a 300 ps long unconstrained molecular dynamics simulation . Calculation of molecular electrostatic potentials revealed that the binding site of OpB is more negative than that of PEP, in accordance with the experimentally observed selectivity of OpB towards proteolysis at dibasic sites . A recently developed Monte Carlo docking method was used provide a structural rationale for the affinity differences measured between Z-Arg and Z-Arg-Arg substrates. Chin J Biotechnol, 1999, 15(3), 159 - 63 Expression of nerve growth factor from Agkistrodon halys Pallas in E . coli and its purification; Guo L et al.; The nerve growth factor (NGF) gene of Agkistrodon halys Pallas was cloned into a secretive prokaryotic expression vector pET-22b+ which carried a C-terminal His . Tag sequence . After transforming into E . coli BL21 (DE3), NGF was induced to express at 30 degrees C by IPTG . SDS-PAGE analysis showed an induced expression product band which constituted about 20% of the total bacterial proteins . However, its molecular weight was larger than what was expected . Moreover, the analysis of product solubility revealed that NGF was in the form of inclusion bodies . The inclusion bodies were solubilized in 6 mumol/L guanidine HCl and purified directly by immobilized metal (Ni2+) chelation affinity chromatography . The product was renatured by dilution and air oxidation in the presence of 5 mumol/L CuSO4, and was proved active by examining the survival rate of PC12 cells in a serum-free medium. Mol Biotechnol, 1999 Dec 15, 13(3), 223 - 45 Recombinant vaccinia viruses . Design, generation, and isolation; Broder CC et al.; The technologies of recombinant gene expression have greatly enhanced the structural and functional analyses of genetic elements and proteins . Vaccinia virus, a large double-stranded DNA virus and the prototypic and best characterized member of the poxvirus family, has been an instrumental tool among these technologies and the recombinant vaccinia virus system has been widely employed to express genes from eukaryotic, prokaryotic, and viral origins . Vaccinia virus is also the prototype live viral vaccine and serves as the basis for well established viral vectors which have been successfully evaluated as human and animal vaccines for infectious diseases and as anticancer vaccines in a variety of animal model systems . Vaccinia virus technology has also been instrumental in a number of unique applications, from the discovery of new viral receptors to the synthesis and assembly of other viruses in culture . Here we provide a simple and detailed outline of the processes involved in the generation of a typical recombinant vaccinia virus, along with an up to date review of relevant literature. J Med Microbiol, 2000 Aug, 49(8), 709 - 12 Bactericidal effect of extracorporeal shock waves on Staphylococcus aureus; von Eiff C et al.; Despite considerable knowledge about the effects of shock waves on eukaryotic soft tissues, no data are available concerning their effect on prokaryotic micro-organisms . In vitro studies on the bactericidal effect of extracorporeal shock waves on staphylococci were performed with energy levels that are standard for the disintegration of calculi . Suspensions containing 10(4)-10(5) cfu of Staphylococcus aureus/ml were sealed in plastic tubes and exposed to shock waves, resulting in a mean decrease of 3.1 log(10) . Whereas impulse rates of > or =350 resulted in a decrease of cfu/ml equalling the detection limit, lower numbers of impulses did not result in an appreciable bactericidal effect . The bactericidal effect of extracorporeal shock waves might provide the basis for the development of novel therapeutic strategies for bacterial infections. Nat Struct Biol, 2000 Aug, 7(8), 626 - 33 A tale of two components: a novel kinase and a regulatory switch; Robinson VL et al.; Histidine protein kinases and response regulators form the basis of phosphotransfer signal transduction pathways . Commonly referred to as two-component systems, these modular and adaptable signaling schemes are prevalent in prokaryotes . Structures of the core domains of histidine kinases reveal a protein kinase fold different from that of the Ser/Thr/Tyr protein kinase family, but similar to that of other ATP binding domains . Recent structure determinations of phosphorylated response regulator domains indicate a conserved mechanism for the propagated conformational change that accompanies phosphorylation of an active site Asp residue . The altered molecular surface promotes specific protein-protein interactions that mediate the downstream response. Microbiology, 2000 Aug, 146 ( Pt 8), 1797 - 814 The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations; Jack DL et al.; In this paper an analysis of 175 currently sequenced transport proteins that comprise the amino acid/polyamine/organocation (APC) superfamily is reported . Members of this superfamily fall into 10 well-defined families that are either prokaryote specific, eukaryote specific or ubiquitous . Most of these proteins exhibit 12 probable transmembrane spanners (TMSs), but members of two of these families deviate from this pattern, exhibiting 10 and 14 TMSs . All members of these families are tabulated, their functional properties are reviewed and phylogenetic/sequence analyses define the evolutionary relationships of the proteins to each other . Evidence is presented that the APC superfamily may include two other currently recognized families that exhibit greater degrees of sequence divergence from APC superfamily members than do the proteins of the 10 established families from each other . At least some of the protein members of these two distantly related families exhibit 11 established TMSs . Altogether, the APC superfamily probably includes 12 currently recognized families with members that exhibit exclusive specificity for amino acids and their derivatives but which can possess 10, 11, 12 or 14 TMSs per polypeptide chain. Mol Microbiol, 2000 Aug, 37(3), 455 - 66 Plasmid and chromosome partitioning: surprises from phylogeny; Gerdes K et al.; Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division . Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes . We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms . All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e . the partition complex) . The proteins are encoded by two genes in an operon that is autoregulated by the par-encoded proteins . In all cases, the upstream gene encodes an ATPase that is essential for partitioning . Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e . the poles or quarter-cell positions) . Two types of partitioning ATPases are known: the Walker-type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin-like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus) . A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid-encoded ATPases clustered into distinct subgroups . Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib . A second surprise was that almost all chromosome-encoded ATPases, including members from both Gram-negative and Gram-positive Bacteria and Archaea, clustered into one distinct subgroup . The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea . Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria . These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci. Mol Microbiol, 2000 Jun, 36(6), 1391 - 402 Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator; Munson GP et al.; Strains of enterotoxigenic Escherichia coli that express CS1 and CS2 pili require the transcriptional activator Rns, a member of the AraC family, for the expression of the pilin genes . Rns is also an activator of its own expression . However, the arrangement of its binding sites near its own promoter is unusual for a prokaryotic activator . Most activators have at least one binding site 30-80 nucleotides upstream of the transcription start site, but Rns has a single upstream binding site centred at -227 . Rns also has two binding sites downstream of the transcription start site centred at +43 and +82, a region generally thought to be reserved for repressors . In vitro, the binding of a MBP::Rns fusion protein to each of these sites facilitates the binding of RNA polymerase to the rns promoter and the formation of an open complex . In vivo, the upstream binding site and one downstream site are required for Rns-dependent activation of its promoter despite the atypical location of these binding sites for an activator . This suggests that Rns may represent a new class of prokaryotic activators. Eur J Biochem, 2000 Aug, 267(16), 4945 - 59 Sequence diversification of the FK506-binding proteins in several different genomes; Galat A; Sequences of FK506-binding proteins (FKBPs) from four genomes of the following organisms were compared: the prokaryote Escherichia coli, the lower eukaryote Saccharomyces cerevisiae, the plant Arabidopsis thaliana, the nematode Caenorhabditis elegans and a composite of 14 unique FKBPs from two mammalian organisms Homo sapiens (man) and Mus musculus (domestic mouse) . A singular FK506-like binding domain (FKBD) has about 12 kDa and occurs in the form of archetypal FKBP-12 and as a part of different proteins ranging in size from 13 to 135 kDa . Some organisms may contain a variable number of proteins which consist from two to four consecutively fused FKBDs . In the 12-kDa subgroup of archetypal FKBPs sequence identity (ID) varies from 100 to 83% (mammalian FKBPs-12), 75-50% in mammalian vs . invertebrate FKBPs-12, and fall to about 30% for pairwise sequence comparisons of mammalian and bacterial FKBPs-12 which suggests that their sequences are divergent . Multiple sequence alignment of FKBPs from the four genomes and a set of unique mammalian FKBPs does not contain any explicit consensus sequence but certain sequence positions have conserved physico-chemical characteristics . Variations of hydrophobicity and bulkiness in the multiple sequence alignment are nonsymmetrical because the physico-chemical properties of the aligned sequences changed during evolution . These variations at the sequence positions which are crucial for binding the immunosuppressive macrolide FK506 and peptidyl-prolyl cis/trans isomerase (PPIase) activity are small. Nucleic Acids Res, 2000 Aug 15, 28(16), 3143 - 50 Recognition of native DNA methylation by the PvuII restriction endonuclease; Rice MR et al.; Recognizing the methylation status of specific DNA sequences is central to the function of many systems in eukaryotes and prokaryotes . Restriction-modification systems have to distinguish between 'self' and 'non-self' DNA and depend on the inability of restriction endonucleases to cleave their DNA substrates when the DNA is appropriately methylated . These endonucleases thus provide a model system for studying the recognition of DNA methylation by proteins . We have characterized the interaction of R.PVU:II with DNA containing the physiologically relevant N4-methylcytosine modification . R.PVU:II binds (N4m)C-modified DNA and cleaves it very slowly . Methylated strands in hemimethylated duplexes were cleaved at a higher rate than in fully methylated duplexes, in parallel with a higher binding affinity for hemimethylated DNA . The co-crystal structures of R.PVU:II-DNA, together with a mutagenesis study, have implicated specific amino acids in recognition of the methylatable base; one of these is His84 . We report that replacing His84 with Ala reduced the rate of cleavage of unmodified DNA but, in contrast, slightly increased the cleavage of (N4m)C-modified DNA. J Mol Biol, 2000 Aug 11, 301(2), 301 - 21 A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments; Morgan DG et al.; Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes . During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum . An understanding of these processes will require the detailed structure of a eukaryotic ribosome . To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution . In general, we find that the active sites for protein synthesis and translocation have been highly conserved . It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove . By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center . In addition, both the small and large subunits are built around a dense tubular network . Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network . We surmise that many of these features correspond to rRNA, based on biochemical and structural data.Ribosomal function is critically dependent on the specific association of small and large subunits . Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes . In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit . Moreover, a novel bridge is formed between the platform and the base of the L1 domain . Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers . Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation . J Mol Biol, 2000 Aug 4, 301(1), 75 - 100 Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes; Paulsen IT et al.; Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms . These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea . Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families . These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied . The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members . The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell . The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes . Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced . Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria . These results provide insight into the relevance of transport to the overall physiology of prokaryotes . Res Microbiol, 2000 Jun, 151(5), 353 - 60 Optimization of green fluorescent protein expression vectors for in vitro and in vivo detection of Listeria monocytogenes; Fortinea N et al.; The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is a useful reporter molecule for monitoring in vivo gene expression in eukaryotic and prokaryotic cells . We constructed a series of GFP vectors for in situ detection of the intracellular pathogen Listeria monocytogenes . The gfp-mutl gene, which encodes a red-shifted GFP, was transcriptionally fused to a strong L . monocytogenes promoter and inserted into various Escherichia coli-Listeria shuttle vectors: i) the integrative monocopy plasmid pAT113; ii) the low copy number plasmid pTCV-Exl; iii) the high copy number plasmid pAT18 . Listeria cells harboring pNF6 and pNF7, constructed from pAT113 and pTCV-Exl, respectively, gave low fluorescence intensities, and were optically detected in cultured macrophages, but not in tissue sections . The fluorescence of Listeria with the pAT18 derivative pNF8 was about 40 times greater than that with pNF6 and 15 times greater than that with pNF7 . Listeria cells harboring pNF8 were readily detected in both cultured macrophages and tissue sections . Constructed GFP vectors did not affect the virulence of L . monocytogenes in a murine model of infection. Genetika, 2000 Jun, 36(6), 758 - 66 {Homologous recombination between direct repeats of chromosomal segments comprising heterozygotic duplications in Escherichia coli}; Sukhodolets VV; In conjugational matings between double mutants for the deo operon of Escherichia coli, haploid recombinants and extended tandem duplications deoC deoD/deoA deoB::Tn5 with the DeoC+DeoA+DeoB+DeoD- phenotype are formed (the deoD+ allele is not expressed due to the polar effect of the Tn5 insertion) . Selection for the expression of the recessive deoC deoD alleles (in the thyA genome) leads to the segregation of haploid clones by duplications and also of clones that retain the diploidy but that are homozygous for deoC deoD . In addition to haploids, diploid clones retaining the duplications have also been found among the DeoD+ segregants . The phenotype of segregants retaining the duplication shows that they were formed by an unequal exchange between sister chromosomes . A comparison of segregation frequency of haploid and diploid DeoD+ clones in rec+ and recBC sbcB sbcC strains shows that duplications in the rec+ genome are more stable . On this basis, it is assumed that the RecBCD pathway possibly makes a greater contribution than the RecF pathway to the preservation of heterozygous duplications playing an important role in the evolution of prokaryotes. FEMS Microbiol Ecol, 2000 Jul 1, 33(1), 81 - 84 Comment to Sherr and Sherr (1999): "Is there any appropriate way to distinguish different beta-N-acetylhexosaminidase activities in aquatic environments?" Vrba J. The recent paper of Sherr and Sherr on detecting low-affinity beta-glucosaminidase activity in several marine microbes extends current knowledge about hydrolytic enzyme activities in natural aquatic systems . However, their conclusions regarding the whole-cell assay with MUF-N-acetyl-beta-D-glucosaminide (MUF-{GlcNAc}) cannot be accepted . First, we explicitly demonstrate a strong correlation between extracellular activities of the high-affinity enzymes and grazing rates of bacterivorous protists . Therefore, the assay can still be recommended for the estimation of total protistan grazing on prokaryotic picoplankton . Second, the ability of many aquatic organisms to produce enzymes which cleave fluorogenic substrates, such as MUF-{GlcNAc} and/or MUF-beta-D-N,N',N"-triacetylchitotriose (MUF-{GlcNAc}(3)), has been well-documented during the last decade . Thus, neither of the two substrates may be considered as exclusively specific for targeting either lysozymes or beta-N-acetylhexosaminidases. Trends Biochem Sci, 2000 Aug, 25(8), 397 - 401 Diversity of transport mechanisms: common structural principles; Driessen AJ et al.; Traditionally, prokaryotic solute transport systems are classified into major groups based on the energetic requirement of the transport process . These include the secondary transporters that are driven by a proton or sodium motive force, and the ATP-binding cassette (ABC) primary transporters, which use the hydrolysis of ATP to fuel transport . These transporters are specified by entirely different architectures of polypeptides . Recently, transport systems have been discovered that are composed of combinations of distinct functional modules of both secondary and ABC transporters . These findings indicate that during evolution the combination of integral membrane transport proteins with either a periplasmic solute-binding protein or a cytosolic ATPase, or both, have resulted in distinct classes of transporters with unique architectures and properties. Mutat Res, 2000 Jun 30, 451(1-2), 91 - 105 Damage-induced recombination in the yeast Saccharomyces cerevisiae; Kupiec M; Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity following DNA damage from endogenous and exogenous genotoxic sources . One of the mechanisms used to repair damaged chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a damaged region of the genome.In this review, I summarized what is known about the molecular and cellular mechanisms by which various DNA-damaging agents induce recombination in yeast . The yeast Saccharomyces cerevisiae has served as an excellent model organism to study the induction of recombination . It has helped to define the basic phenomenology and to isolate the genes involved in the process . Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans . Many carcinogens are known to induce recombination and to cause chromosomal rearrangements . An understanding of the mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans. J Biol Chem, 2000 Oct 6, 275(40), 31024 - 9 Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR; Outten FW et al.; Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced . Depending upon the cellular copper status, copper ions must either be imported or ejected . CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown . Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family . These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase . Homologues of YacK are found in copper resistance determinants that facilitate copper efflux . Here we show by targeted gene deletion and promoter fusion assays that both copA and yacK are regulated in a copper-responsive manner by the MerR homologue, ybbI . We have designated ybbI as cueR for the Cu efflux regulator . This represents the first example of a copper-responsive regulon on the E . coli chromosome and further extends the roles of MerR family members in prokaryotic stress response. Proc Natl Acad Sci U S A, 2000 Aug 1, 97(16), 8944 - 9 Trans catalysis in Tn5 transposition; Naumann TA et al.; Synaptic complexes in prokaryotic transposons occur when transposase monomers bind to each of two specific end-binding sequences and then associate to bring the proteins and the two ends of the transposon together . It is within this complex of proteins and DNA that identical catalytic reactions are carried out by transposase on each of the ends of the transposon . In this study, we perform in vitro transposition reactions by combining the methylated inside end (IE(ME)) biased hyperactive Tn5 transposase, Tnp sC7 version 2.0, and the outside end (OE) biased hyperactive Tn5 transposase, Tnp EK/LP, with plasmid DNA containing a transposon defined by one IE(ME) and one OE . These two proteins cooperate to facilitate double end cleavage of the transposon from the plasmid and conversion into transposition products via strand transfer . When one of the hyperactive Tnps is replaced with a catalytically inactive version containing the mutation EA326 (DDE mutant), the predominant reaction product is a linearized plasmid resulting from single end cleavage . Restriction analysis of these linear products reveals that cleavage is occurring on the end distal to that which is bound by the transposase with an intact active site or in trans . Similar in vitro experiments performed with precut transposons and a supercoiled target plasmid demonstrated that the strand transfer reaction is also facilitated by a trans active DDE motif. Semin Cell Dev Biol, 2000 Jun, 11(3), 149 - 58 Getting in and out of the proteasome; Glickman MH; By far the best understood role of the proteasome is to remove ubiquitin-conjugated proteins from eukaryotric cells by hydrolysing them into small peptides of varying lengths . These include both misfolded/abnormal proteins, as well as 'normal' proteins that need to be rapidly removed for regulatory purposes . However, the proteasome is also present in numerous prokaryotic organisms, while ubiquitin, and the ubiquitin conjugating system, are not . The eukaryotic proteasome has been adapted to degrading proteins in a ubiquitin-dependent fashion by the addition of regulatory factors that assemble in different layers onto the proteolytic core of the proteasome, and by increasing the diversity of the core subunits as well . In addition to hydrolysing ubiquitinated proteins into amino acids, the proteasome can also proteolyse selected non-ubiquitinated proteins, process proteins, and possibly refold misfolded proteins . This review will focus on the different proteasome functions, and how these are used in the multiple regulatory roles the proteasome plays in eukaryotic cells. Insect Mol Biol, 2000 Jun, 9(3), 315 - 22 The highly compact structure of the mitochondrial DNA polymerase genomic region of Drosophila melanogaster: functional and evolutionary implications; Lefai E et al.; The structure of a Drosophila melanogaster genomic region containing five tightly clustered genes has been determined and evaluated with regard to its functional and evolutionary relationships . In addition to the genes encoding the two subunits (alpha and beta) of the DNA polymerase gamma holoenzyme, the key enzyme for mitochondrial DNA replication, other genes contained in the cluster may be also involved in the cellular distribution of mitochondria and in the coordination of mitochondrial and nuclear DNA replication . The gene cluster is extremely compact, with very little intergenic space . It contains two bidirectional promoter regions, and particularly notable is the 5' end overlap detected in two of its genes, an exceptional situation in both prokaryotic and eukaryotic genome organization. DNA Seq, 2000, 11(1-2), 119 - 24 A DNA fragment of Desulfovibrio gigas genome containing replication origin related genes; Broco M et al.; The nucleotide sequence of a 10,772 base pair (bp) region from Desulfovibrio gigas genome was determined . This sequence, which is adjacent to the region containing the coding units for the metalloproteins rubredoxin-oxygen oxidoreductase (ROO) and rubredoxin, includes the flavodoxin gene . Additionally, it also contains four open reading frames (ORFs) related to genes frequently found in replication origin regions of prokaryotes . These hypothetical encoded polypeptides are: the response regulator proteins (PhoP and PhoR) from the phosphate regulon, a DNA partitioning protein and an asparagine synthetase. IUBMB Life, 2000 May, 49(5), 411 - 20 Selenium, the element of the moon, in life on earth; Flohe L et al.; The present status of selenium biochemistry is reviewed with particular emphasis on biomedical problems related to the selenium status of humans and experimental animals . Historical milestones of selenium biochemistry starting from the identification of the first selenoenzymes up to the elucidation of prokaryotic and eukaryotic selenoprotein biosynthesis are compiled . Topical hypotheses on the biological role of selenium in general and of individual selenoproteins in respect to antioxidant defense, redox regulation of metabolic processes, thyroid function, spermatogenesis, oncogenesis, and atherogenesis are critically evaluated. Anticancer Drug Des, 2000 Apr, 15(2), 151 - 60 In vitro characterization of the anticancer activity of membrane-active cationic peptides . I . Peptide-mediated cytotoxicity and peptide-enhanced cytotoxic activity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines; Johnstone SA et al.; Cationic amphipathic peptides, such as the defensins and cecropins, induce cell death in prokaryotic and eukaryotic cells by increasing membrane permeability . Increased permeability may lead to cell lysis or, alternatively, may produce subtle changes in the membrane's barrier function that promote cell death . The in vitro cytotoxic and lytic activity of short mammalian-derived extended-helical cationic peptides and insect-derived alpha-helical peptides was measured in this study with the objective of establishing the anticancer potential of these agents . Two specific aims were addressed: (i) to assess the activity of peptides against non-malignant cells (sheep erythrocytes and human umbilical vein endothelial cells) versus tumor cells; and (ii) to characterize the cytotoxic activity using multidrug-resistant tumor cell lines in the presence and absence of the anthracycline doxorubicin . Cell lysis assays demonstrated that the lytic activity of the peptides tested was 2->50 times more cytotoxic to tumor cells than to non-malignant cells . Further, the cytotoxic activity of these peptides was equivalent when tested against sensitive and multidrug-resistant cell lines . In addition to their inherent cytotoxic activity, these membrane-active peptides can also augment the in vitro cytotoxic activity of doxorubicin against multidrug-resistant tumor cells. Bioorg Khim, 2000 May, 26(5), 340 - 51 {The endogenous differentiation factor of the HL-60 cells shows a nuclease activity}; Dranitsyna SM et al.; A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of the hldf gene in prokaryotic systems was studied . On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified . The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage lambda, and all forms of plasmid DNA . We established that treatment of the HL-60 cell culture with this peptide (10(-6) M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis. Micron, 2001 Jan, 32(1), 43 - 50 Structural comparison of prokaryotic and eukaryotic chaperonins; Carrascosa JL et al.; Chaperonins are key components of the cell machinery and are involved in the productive folding of proteins . Most chaperonins share a common general morphology based in a cylinder composed of two rings of 7-9 subunits, with a conspicuous cavity inside the particle . Chaperonins have been classified into two groups according to their sequence homologies: type I, whose better known member is GroEL, and type II comprising the eukaryotic cytosolic CCT and the archaebacterial thermosome, among others . Although the basic structure of both chaperonin types is rather similar, there are a number of basic differences among them . Whereas GroEL is rather non-specific regarding its substrate, CCT is more specialized, and plays a fundamental role in the folding of cytoskeletal proteins . Another important difference is that GroEL is an homopolymer, while CCT is an heteromeric complex built up of eight different polypeptides . Furthermore, GroEL requires a cofactor (GroES) that is not present in the type II chaperonins.Recent studies of the structure of CCT have allowed a deeper insight into its function . Electron microscopic analyses have revealed a different behavior of this chaperonin after binding to nucleotides, respect to GroEL . The atomic structure of the thermosome fits into the electron microscopy reconstructed volume of the CCT . This fitting gives clues to compare the structural transitions of GroEL and CCT during the folding cycle . The different changes undergone by the two chaperonins suggest the existence of differences in the way they bind substrates and enlarge the internal cavity, as well as a different type of signaling between the two rings of the types I and II chaperonins. J Biol Chem, 2000 Oct 6, 275(40), 30753 - 6 Crystal structure of human frataxin; Dhe-Paganon S et al.; Friedreich's ataxia, an autosomal recessive neurodegenerative disorder characterized by progressive gait and limb ataxia, cardiomyopathy, and diabetes mellitus, is caused by decreased frataxin production or function . The structure of human frataxin, which we have determined at 1.8-A resolution, reveals a novel protein fold . A five-stranded, antiparallel beta sheet provides a flat platform, which supports a pair of parallel alpha helices, to form a compact alphabeta sandwich . A cluster of 12 acidic residues from the first helix and the first strand of the large sheet form a contiguous anionic surface on the protein . The overall protein structure and the anionic patch are conserved in eukaryotes, including animals, plants, and yeast, and in prokaryotes . Additional conserved residues create an extended 1008-A(2) patch on a distinct surface of the protein . Side chains of disease-associated mutations either contribute to the anionic patch, help create the second conserved surface, or point toward frataxin's hydrophobic core . These structural findings predict potential modes of protein-protein and protein-iron binding. Genes Dev, 2000 Jul 15, 14(14), 1789 - 96 Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing; Qin Z et al.; Long inverted repeats (palindromes) are ubiquitous among prokaryotic and eukaryotic genomes . Earlier work has implicated both DNA breaks and short inverted repeats (IRs) in the formation of long palindromes in yeast and Tetrahymena by a proposed mechanism of intramolecular recombination . Here we report that long-palindromic linear plasmids are formed in Streptomyces following double strand DNA breakage by a nonrecombinational intra-strand annealing process that also involves IRs . By modification of palindrome-generating linear plasmids and development of a novel procedure that enables the sequencing of palindrome junctions, we show that long-palindrome formation occurs by unimolecular intra-strand annealing of IRs followed by 3' extension of the resulting DNA fold-back . The consequent hairpin structures serve as templates for synthesis of duplex linear plasmids containing long palindromes . We suggest that this model for long-palindrome formation in Streptomyces may represent a generally applicable mechanism for generating DNA palindromes. J Biomol Screen, 2000 Jun, 5(3), 141 - 52 Novel fluorescent technology platform for high throughput cytotoxicity and proliferation assays; Wodnicka M et al.; We have developed a novel fluorescent Oxygen BioSensor technology platform adaptable to many applications in the area of drug discovery and development, particularly cell-based assays . This biosensor technology requires no additional reagents or incubations, and affords continuous real-time readout of dissolved oxygen concentrations . Since the level of oxygen dissolved in an assay's medium correlates to the number and viability of the cells in the medium, this technology is ideally suited for monitoring cell viability, proliferation, or death . The technology is particularly well suited to investigating cells' kinetic responses to proliferative or toxic stimuli, such as drugs . When incorporated into a 96- or 384-well microplate format, it is compatible with standard laboratory automation systems . Here we present data illustrating the application of the Oxygen BioSensor technology for rapid, homogeneous detection and evaluation of metabolic activity of a variety of eukaryotic and prokaryotic cells, including mammalian cells, insect cells, yeast, and bacteria . In the absence of toxic substances, we find a good correlation between cell number and signal over a wide range of cell concentrations and growth times . To evaluate the usefulness of the Oxygen BioSensor for cytotoxicity assays, we have performed a series of experiments using a range of toxic agents and cell types, including both bacteria and mammalian cell lines . In a side-by-side comparison to standard MTT assays using HL60 cells, comparable IC(50) values were found with the Oxygen BioSensor for five different toxins or drugs . This assay method does not have the need for additional reagents, handling steps, or incubation periods required by the MTT assays. J Bacteriol, 2000 Aug, 182(15), 4268 - 77 A redox-responsive regulator of photosynthesis gene expression in the cyanobacterium Synechocystis sp . Strain PCC 6803; Li H et al.; We have identified genes in the unicellular cyanobacterium Synechocystis sp . strain PCC 6803 that are involved with redox control of photosynthesis and pigment-related genes . The genes, rppA (sll0797) and rppB (sll0798), represent a two-component regulatory system that controls the synthesis of photosystem II (PSII) and PSI genes, in addition to photopigment-related genes . rppA (regulator of photosynthesis- and photopigment-related gene expression) and rppB exhibit strong sequence similarity to prokaryotic response regulators and histidine kinases, respectively . In the wild type, the steady-state mRNA levels of PSII reaction center genes increased when the plastoquinone (PQ) pool was oxidized and decreased when the PQ pool was reduced, whereas transcription of the PSI reaction center genes was affected in an opposite fashion . Such results suggested that the redox poise of the PQ pool is critical for regulation of the photosystem reaction center genes . In Delta rppA, an insertion mutation of rppA, the PSII gene transcripts were highly up-regulated relative to the wild type under all redox conditions, whereas transcription of phycobilisome-related genes and PSI genes was decreased . The higher transcription of the psbA gene in Delta rppA was manifest by higher translation of the D1 protein and a concomitant increase in O(2) evolution . The results demonstrated that RppA is a regulator of photosynthesis- and photopigment-related gene expression, is involved in the establishment of the appropriate stoichiometry between the photosystems, and can sense changes in the PQ redox poise. Am J Physiol Endocrinol Metab, 2000 Jul, 279(1), E116 - 23 Biological activities of recombinant chicken leptin C4S analog compared with unmodified leptins; Dridi S et al.; The chicken leptin sequence, in contrast to mammalian leptins, contains an unpaired Cys at position 3 of the original cDNA (AF012727) . The presence of an extra Cys may confer a different structure and affect the leptin's biological activity . To address this, we studied the effects of wild-type and mutated (C4S) chicken leptins in vitro and in vivo and compared them with mammalian leptin prepared from ovine leptin cDNA . The prokaryotic expression vector pMON, encoding full-size A(-1) chicken leptin (AF012727), was mutated using a mutagenesis kit, yielding the C4S analog . Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin C4S upon induction with nalidixic acid . The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding three electrophoretically pure fractions, eluted from the column by 100, 125, and 150 mM NaCl, respectively . All three fractions showed a single band of the expected molecular mass (16 kDa) and were composed of >95% monomeric protein . Proper refolding was evidenced by comparing the circular dichroism spectrum of the analog with spectra of nonmutated chicken and ovine leptins . The biological activity of the C4S analog was evidenced by its ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct similar to its nonmutated counterpart, indicating that Cys4 plays no role in leptin activity . The in vitro activity of both wild-type and mutated chicken leptins was approximately 10-fold lower than that of ovine leptin . After intravenous or intraperitoneal injections, C4S analog and the nonmutated chicken and ovine leptins all lowered the food intake of starved 9-day-old broiler or 5-wk-old layer male chickens by 11-34% . Monitoring food behavior revealed that the attenuated food intake resulted not from a decreased number of approaches to the feeders but from a decrease in the average time spent eating during each approach. Biochemistry, 2000 Jul 11, 39(27), 7973 - 83 Kinetic and mechanistic studies of signal peptidase I from Escherichia coli; Stein RL et al.; Signal peptidases of prokaryotic organisms reside in the outer leaflet of the cytoplasmic membrane and catalyze the hydrolytic cleavage of a specific peptide bond of membrane-imbedded preproteins to liberate mature proteins for secretion . In this manuscript, we report new and efficient peptide substrates for SPase and their use to explore features of this enzyme's reaction mechanism . The enzyme used in this study was recombinant SPase I of Escherichia coli that had been solubilized with Triton X-100 and purified to near homogeneity . Our new substrates are based on the fluorogenic peptide reported by Zhong and Benkovic {(1998) Anal . Biochem . 255, 66}, Y(NO2)FSASALA approximately KIK(Abz)-NH(2) (Y(NO2), 3-nitro-L-tyrosine; K(Abz), epsilon-(2-aminobenzoyl)-L-Lys; hydrolysis at A approximately K) . We found that when a signal peptide-like sequence is appended onto the N-terminus of this peptide to produce K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2), k(c)/K(m) increases from 85 to 2.5 x 10(6) M(-)(1) s(-)(1) . k(c)/K(m) decreases with increasing concentration of Triton X-100 micelles under the condition {Triton X-100}(micelle) > {S}(0) > {E}(0) . We explain this apparent inhibition with a model of surface dilution kinetics in which "empty" micelles compete with substrate-containing micelles for micelle-bound enzyme . Fusion of micelle-bound enzyme with a substrate-containing micelle leads to formation of productive E:S substrate complexes while fusion of micelle-bound enzyme with an "empty" micelle is nonproductive and inhibitory . The dependence of steady-state kinetic parameters for the SPase-catalyzed hydrolysis of K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2) on {Triton X-100}(micelle) supports this model . Product inhibition and solvent isotope effects were also investigated and could be interpreted in the context of this model. Biochemistry, 2000 Jul 11, 39(27), 7856 - 62 IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of {2Fe-2S} and {4Fe-4S} clusters in IscU; Agar JN et al.; Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes . The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide . In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography . The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and Mossbauer investigations . The results show sequential cluster assembly with the initial IscU product containing one {2Fe-2S}(2+) cluster per dimer converting first to a form containing two {2Fe-2S}(2+) clusters per dimer and finally to a form that contains one {4Fe-4S}(2+) cluster per dimer . Both the {2Fe-2S}(2+) and {4Fe-4S}(2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air . On the basis of sequence considerations and spectroscopic studies, the {2Fe-2S}(2+) clusters in IscU are shown to have incomplete cysteinyl ligation . In addition, the resonance Raman spectrum of the {4Fe-4S}(2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site . The ability to assemble both {2Fe-2S}(2+) and {4Fe-4S}(2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins. Proc Natl Acad Sci U S A, 2000 Jul 18, 97(15), 8257 - 62 Crystal structure of the holliday junction DNA in complex with a single RuvA tetramer; Ariyoshi M et al.; In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins . Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration . We solved the crystal structure of the Holliday junction bound to a single Escherichia coli RuvA tetramer at 3.1-A resolution . In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center . The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry . Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit . The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding. Gen Comp Endocrinol, 2000 May, 118(2), 302 - 9 Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): subcloning, preparation, and characterization; Sandowski Y et al.; The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid . The bacterially expressed trPRLR-ECD protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl . The bioactive monomeric 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture . The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column . Binding experiments using {125I}ovine placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nM . Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM . Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL . Only oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes, with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or trPRLR-ECD:oPRL ratios . No studies were performed with chPRL because of its inability to compete with {125I}oPL or with trPRL because of its low affinity toward trPRLR-ECD . The present results agree with previous findings indicating, as in mammals, that homologous PRL interacts transiently with its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected. J Virol, 2000 Aug, 74(15), 6784 - 9 Evidence for nucleic acid binding ability and nucleosome association of Bombyx mori nucleopolyhedrovirus BRO proteins; Zemskov EA et al.; The Bombyx mori nucleopolyhedrovirus (BmNPV) genome contains five related members of the bro gene family, all of which are actively expressed in infected BmN cells . Although their functions are unknown, their amino acid sequences contain a motif found in all known viral and prokaryotic single-stranded DNA binding proteins . To determine if they bind to nucleic acids, we fractionated the nuclei of BmNPV-infected BmN cells using a histone extraction protocol . We detected BRO-A, BRO-C, and BRO-D in the histone H1 fraction using anti-BRO antibodies . Micrococcal nuclease treatment released these BRO proteins from the chromatin fraction, suggesting their involvement in nucleosome structures . Chromatographic fractionation showed that BRO-A and/or BRO-C interacted with core histones . Expression of partial sequences of BRO-A proved that the N-terminal 80 amino acid residues were required for DNA binding activity . We also demonstrated that BmNPV BRO proteins underwent phosphorylation and ubiquitination followed by proteasome degradation, which may explain their distribution in the cytoplasm as well as the nucleus . We propose that BRO-A and BRO-C may function as DNA binding proteins that influence host DNA replication and/or transcription. Biosci Rep, 1999 Apr, 19(2), 73 - 9 The histidine decarboxylase (HDC) gene of Tetrahymena pyriformis is similar to the mammalian one . A study of HDC expression; Hegyesi H et al.; RNA was isolated from Tetrahymena pyriformis GL and using human histidine decarboxylase (HDC) gene primers, the RT-PCR product was sequenced . A fraction containing 207 base pairs was compared to the published sequences of prokaryotic and mammalian (rat, mouse and human) HDC cDNA (exons) . The HDC-cDNA fraction of Tetrahymena was similar to the mammalian cDNA-s and it was completely different from the prokaryotic HDC-gene . The results indicate the presence of a mammalian-like HDC-gene already in a unicellular eukaryote organism and demonstrates also that the divergence of the prokaryotic-eukaryotic common gene took place already at this low evolutionary level. J Biol Chem, 2000 Sep 22, 275(38), 29772 - 8 Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo; Tzivion G et al.; 14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein . This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control . We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin . Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3 . The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96) . Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide . The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro . Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro . Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3. Mol Ecol, 2000 Jul, 9(7), 935 - 48 Microdiversity of uncultured marine prokaryotes: the SAR11 cluster and the marine Archaea of Group I; Garcia-Martinez J et al.; The SAR11 cluster and the Group I of marine Archaea represent probably the best two examples of uncultured marine prokaryotes of widespread occurrence . To study their microdiversity and distribution, a total of 81 and 48 clones, respectively, were sequenced from Mediterranean and Antarctic waters at different locations and depths . The DNA regions chosen for the analysis were the last third, approximately, of the 16S rRNA gene and the 16S-23S intergenic spacer (also known as internal transcribed spacer {ITS}) . There was a high concordance in both, even with the extremely variable ITS, where potential probes have been proposed for the identification and isolation of these micro-organisms . In terms of community structure, our results show that although depth-related factors seem to be predominant in the final associations of the clones, geography also plays a significant role . A major group of surface-associated sequences was found in both SAR11 and marine Archaea . In both cases this group was relatively homogeneous containing little diversity in terms of sequence, while sequences retrieved from deep samples and some surface clones contained much more heterogeneity . As a whole, both groups of prokaryotes seem to fall within the limits of well-defined taxonomic units. J Biol Rhythms, 2000 Jun, 15(3), 218 - 24 Rhythmic activity of uptake hydrogenase in the prokaryote Rhodospirillum rubrum; Van Praag E et al.; Growth of Rhodospirillum rubrum was followed in cultures kept under anoxic conditions at constant temperature in either continuous light (LL, 32 degrees C) or continuous darkness (DD, 32 degrees C and 16 degrees C) . In DD, only small modifications of the turbidity were detected; linear regression analysis nevertheless gives a very significant slope (t(34) = 13.07, p < 10(-14), with R2 of 0.834) . Mean generation times reflected these differences of growth with 11.9+/-0.5 h in LL and 43.2+/-1.1 h in DD at 32 degrees C and 37.4+/-1.0 h at 16 degrees C cultures . The uptake hydrogenase (Hup) activity has been followed in situ in whole cells of R . rubrum grown in the same conditions, and a clear ultradian rhythm of activity has been observed . Indeed, after about 12 h in the new media, a rapid rise of hydrogenase activity was observed in both LL and DD cultures after which it decreased again to very low values . The activity of Hup continued to show such fluctuations during the rest of the experiment, both in DD and in LL, during the growth and stationary phases . The Lomb-Scargle power periodogram method demonstrates the presence of a clear rhythmic Hup activity both in LL and DD . In the LL-grown cultures, the oscillating activity is faster and continues throughout the growth and the stationary phases, with an ultradian period of 12.1+/-0.5 h . In DD, the slow-growing bacteria showed an ultradian oscillatory pattern of Hup activity with periods of 15.2+/-0.5 h at 32 degrees C and 23.4+/-2.0 h at 16 degrees C . The different periods obtained for LL- and DD-grown bacteria are significantly different. Science, 2000 Jul 7, 289(5476), 77 - 85 Three-dimensional structure of the Tn5 synaptic complex transposition intermediate; Davies DR et al.; Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome . Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase . Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition . We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution . The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites . This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA. Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 27 - 30 {Functional and structural analysis of a prokaryotic enhancer-like element in Escherichia coli MC1061 strain}; Zhu M et al.; Cat and lacZ genes were used as reporter gene and three prokaryotic enhancer-like element (MC2, MC8 and MC9) were identified in the genomic DNA of MC1061 strain . All three fragments can improve the expression of lacZ gene by 2-5 times with the orientation independence . The results of in vivo transcription and Dot blot hybridization assays suggested that MC8 regulated the expression of lacZ at transcription level . Stepwise deletion expreriments showed the functional domain of MC8 located at 450-950 bp, and in regions 450-600 bp and 840-950 bp contain at least one functional loci . Sequence data indicated three are 3 A + T rich sections in MC8, 2 of them are in the functional loci. Sheng Wu Gong Cheng Xue Bao, 2000 Jan, 16(1), 13 - 6 {Immunization of mice with plasmid DNA against malaria and regulation of antigen expression by tetracycline-controlled promoter}; Xie WK et al.; Sequence of MSP1-31 of Plasmodium falciparum was constructed into eukaryotic expression vector pTRE, which could be repressed by tetracycline (Tc) and resulted in recombinant plasmid pTRE-31 . The plasmid was injected into the quadriceps muscle of BALB/c mice with Tc responsive plasmid pTet-off to measure specific antibodies . The MSP1-31 prokaryotic expressed protein was used as antigen in ELISA . Results showed that mice orally administered by Tc had a seroconversion rate of 7.1% (1/14) 4 weeks after injection, whereas the control mice had a seroconversion rate of 100% and the titers of antibody were raised continusly within 12 weeks . The study suggested that the recombinant plasmids pTRE-31/pTet-off could efficiently induce humoral response against MSP1-31 of malaria . Moreover this immune response was controlled by Tc and was reversible after withdrawal of Tc dilivery . The induction of antibody by removing Tc at the fourth week after injection indicated that DNA vaccine could remain in mice and capable of expressing antigen for at least 4 weeks. Clin Diagn Lab Immunol, 2000 Jul, 7(4), 588 - 95 Antigenic and genetic characterization of lipoprotein LppQ from Mycoplasma mycoides subsp . mycoides SC; Abdo EM et al.; Lipoprotein LppQ, a predominant 48-kDa antigen, and its corresponding gene, lppQ, were characterized in Mycoplasma mycoides subsp . mycoides SC, the etiological agent of contagious bovine pleuropneumonia . The lppQ gene is specific to M . mycoides subsp . mycoides SC and was found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccinal strains . LppQ is encoded as a precursor with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site . The leader sequence shows significant prominent transmembrane helix structure with a predicted outside-to-inside helix formation capacity . The N-terminal domain of the mature LppQ was shown to be surface exposed . It induced a strong, specific, early, and persistent immune response in naturally and experimentally infected animals . The C-terminal domain of LppQ possesses an integral membrane structure built up of repeated units, rich in hydrophobic and aromatic amino acids, which have a pore formation potential . A recombinant peptide representing the N-terminal domain of LppQ was obtained by site-directed mutagenesis of nine Mycoplasma-specific TGA (Trp) codons into universal TGG (Trp) codons and expression in Escherichia coli hosts . It was used for serodetection of cattle infected with M . mycoides subsp . mycoides SC, in which it was detected postinfection for significantly longer than conventional serological test reactions. Arch Virol, 2000, 145(5), 945 - 56 Identification and in vivo expression of a prokaryotic-like ribosome recognition sequence upstream of the coat protein gene of potato virus X; Hefferon KL et al.; Conserved prokaryotic sequence motifs, distinct from the classic Shine-Dalgarno sequence, yet possessing homology to 16S rRNA in E . coli have been identified in a number of plant viruses . In this report, a similar Shine-Dalgarno-like motif located immediately upstream to the CP gene of potato virus X (PVX) was demonstrated to enable expression of a reporter gene in E . coli to approximately one third the level of a similar construct containing the classical Shine-Dalgarno sequence . Both PVX-specific CP and RNA transcripts were detected in chloroplasts purified from transgenic potato plants containing the PVX CP gene and corresponding leader sequence . Protoplasts generated from these transgenic plants were used to demonstrate that expression of the PVX CP from chloroplasts is possible . The implications of these results on the PVX infection cycle are discussed. Nat Struct Biol, 2000 Jun, 7(6), 475 - 8 The crystal structure of NusB from Mycobacterium tuberculosis; Gopal B et al.; Both prokaryotes and eukaryotes regulate transcription through mechanisms that suppress termination signals . An antitermination mechanism was first characterized in bacteriophage lambda . Bacteria have analogous machinery that regulates ribosomal RNA transcription and employs host factors, called the N-utilizing (where N stands for the phage lambda N protein) substances (Nus), NusA, NusB, NusE and NusG . Here we report the crystal structure of NusB from Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans . This molecule shares a similar tertiary structure with the related Escherichia coli protein but adopts a different quaternary organization . We show that, unlike the E . coli homolog, M . tuberculosis NusB is dimeric both in solution and in the crystal . These data help provide a framework for understanding the structural and biological function of NusB in the prokaryotic transcriptional antitermination complex. Infect Control Hosp Epidemiol, 2000 Jun, 21(6), 404 - 10 Toward new biomaterials; Montdargent B et al.; Polymers are widely used for a large range of medical devices used as biomaterials on a temporary, intermittent, and long-term basis . It is now well accepted that the initial rapid adsorption of proteins to polymeric surfaces affects the performance of these biomaterials . However, protein adsorption to a polymer surface can be modulated by an appropriate design of the interface . Extensive study has shown that these interactions can be minimized by coating with a highly hydrated layer (hydrogel), by grafting on the surface different biomolecules, or by creating domains with chemical functions (charges, hydrophilic groups) . Our laboratory has investigated the latter approach over the past 2 decades, in particular the synthesis and the biological activities of polymers to improve the biocompatibility of blood-contacting devices . These soluble and insoluble polymers were obtained by chemical substitution of macromolecular chains with suitable groups able to develop specific interactions with biological components . Applied to compatibility with the blood and the immune systems, this concept has been extended to interactions of polymeric biomaterials with eukaryotic and prokaryotic cells . The design of new biomaterials with low bacterial attachment is thus under intensive study . After a brief overview of current trends in the surface modifications of biocompatible materials, we will describe how biospecific polymers can be obtained and review our recent results on the inhibition of bacterial adhesion using one type of functionalized polymer obtained by random substitution . This strategy, applied to existing or new materials, seems promising for the limitation of biomaterial-associated infections. Infect Control Hosp Epidemiol, 2000 Jun, 21(6), 390 - 3 Potential dissemination of antibiotic resistance genes from transgenic plants to microorganisms; Bertolla F et al.; Evidence that genes were transferred during evolution from plants to bacteria was obtained from nucleotide and protein sequence analyses . However, the extent of such transfers among phylogenetically distant organisms is limited by various factors, including those related to complexity of the environment and those endogenous to the bacteria, designed to prevent a drift of the genome integrity . The goal of this article is to give an overview of the potentials and limits of natural interkingdom gene transfers, with a particular focus on prokaryote originating sequences fitting the nuclear genome of transgenic plants. Trends Microbiol, 2000 Jul, 8(7), 313 - 20 Plasmid and chromosome segregation in prokaryotes; Moller-Jensen J et al.; Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components . Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic-like apparatus in prokaryotes . The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Bioessays, 2000 Jul, 22(7), 666 - 72 Actin-related proteins (Arps): conformational switches for chromatin-remodeling machines? Boyer LA, Peterson CL. The actin superfamily of ATPases includes cytoskeletal actins, the stress 70 proteins (e.g . hsc70), sugar kinases, glycerol kinase, and several prokaryotic cell cycle proteins . Although these proteins share limited sequence identity, they all appear to maintain a similar tertiary structure, the "actin fold", which may serve to couple ATP hydrolysis to protein conformational changes . Recently, an actin-related protein (Arp) subfamily has been identified based on sequence homology to conventional actin . Although some Arps are clearly involved in cytoskeletal functions, both actin and/or Arps have been found as stoichiometric subunits of several nuclear chromatin-remodeling enzymes . Here we present two related models in which actin and/or Arps function as conformational switches that control either the activity or the assembly of chromatin-remodeling machines. J Biol Chem, 2000 Sep 29, 275(39), 30058 - 63 Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II; Wastl J et al.; We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules . The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor . Rubredoxin was identified in all tested phototrophic eukaryotes . Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane . Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements . The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins . Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway. J Biol Chem, 2000 Sep 8, 275(36), 27851 - 7 The effect of O6-methylguanine DNA adducts on the adenosine nucleotide switch functions of hMSH2-hMSH6 and hMSH2-hMSH3; Berardini M et al.; The human homologs of prokaryotic mismatch repair have been shown to mediate the toxicity of certain DNA damaging agents; cells deficient in the mismatch repair pathway exhibit resistance to the killing effects of several of these agents . Although previous studies have suggested that the human MutS homologs, hMSH2-hMSH6, bind to DNA containing a variety of DNA adducts, as well as mispaired nucleotides, a number of studies have suggested that DNA binding does not correlate with repair activity . In contrast, the ability to process adenosine nucleotides by MutS homologs appears to be fundamentally linked to repair activity . In this study, oligonucleotides containing a single well defined O(6)-methylguanine adduct were used to examine the extent of lesion-provoked DNA binding, single-step ADP --> ATP exchange, and steady-state ATPase activity by hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers . Interestingly, O(6)-methylguanine lesions when paired with either a C or T were found to stimulate ADP --> ATP exchange, as well as the ATPase activity of purified hMSH2-hMSH6, whereas there was no significant stimulation of hMSH2-hMSH3 . These results suggest that O(6)-methylguanine uniquely activates the molecular switch functions of hMSH2-hMSH6. Appl Environ Microbiol, 2000 Jul, 66(7), 2898 - 905 Cospeciation of psyllids and their primary prokaryotic endosymbionts; Thao ML et al.; Psyllids are plant sap-feeding insects that harbor prokaryotic endosymbionts in specialized cells within the body cavity . Four-kilobase DNA fragments containing 16S and 23S ribosomal DNA (rDNA) were amplified from the primary (P) endosymbiont of 32 species of psyllids representing three psyllid families and eight subfamilies . In addition, 0.54-kb fragments of the psyllid nuclear gene wingless were also amplified from 26 species . Phylogenetic trees derived from 16S-23S rDNA and from the host wingless gene are very similar, and tests of compatibility of the data sets show no significant conflict between host and endosymbiont phylogenies . This result is consistent with a single infection of a shared psyllid ancestor and subsequent cospeciation of the host and the endosymbiont . In addition, the phylogenies based on DNA sequences generally agreed with psyllid taxonomy based on morphology . The 3' end of the 16S rDNA of the P endosymbionts differs from that of other members of