Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 186 - 90 {Preparation and characterization of monoclonal antibody against N-terminal domain of polycystin 1}; Zhao HD et al.; AIM: To prepare and identify monoclonal antibody (mAb) against N-terminal domain of polycystin 1 . METHODS: Total RNA was extracted from kidney tissue of a healthy man . Gene sequence encoding polycystin 1 N-terminal domain was amplified by one-step RT-PCR . The target gene was inserted into prokaryotic expression vector pQE30 and transformed into competent cells E . coli M15 . The fusion protein was expressed under IPTG induction and purified by affinity chromatography . The purified fusion protein was then used to immunize BALB/c mice . The splenocytes from immunized mice were fused with myeloma cells Sp2/0 by PEG 4000 mediator method and the hybridomas were selected in HAT medium . The hybridoma clones secreting mAb against polycystin 1 amino-terminal domain were detected by ELISA and cloned by limiting dilution . The specificity of mAb against polycystin 1 N-terminal domain was verified by ELISA and Western blot . RESULTS: cDNA encoding polycystin 1 extracellular region was obtained . Fusion protein of polycystin 1 N-terminal domain were expressed in pQE30 expression system . The relative molecular masses (Mr) of the two fusion proteins were 19,800 and 18,900, respectively . One hybridoma cell 7B1 secreting specific mAb was obtained . Western blot analysis showed that the mAb reacted strongly and specifically to polycystin 1 N-terminal domain . CONCLUSION: polycystin 1 N-terminal fusion proteins have been expressed in E.coli M15 . Anti-fusion protein mAb with antigen-binding activity has been prepared successfully. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 168 - 70 {Expression of Lagurus ZP3 fusion protein in prokaryotic cells and preparation of its antiserum}; Zhang AL et al.; AIM: To express and purify Lugurus zone pellucida 3 (LZP3) in prokaryotic cells and to prepare the LZP3-specific rabbit antiserum . METHODS: The core fragment of LZP3 gene was cloned into plasmid pGEX-4T-1 containing glutathione s-transferase (GST) fusion protein gene . Following restriction enzyme digestion analysis and sequencing, pGEX-4T-LZP3 was transformed into E . coli BL21(DE3) . GST-LZP3 fusion protein was expressed under IPTG induction and further purified with Glutathione Sepharose 4B . Then the purified GST-LZP3 fusion protein was used to immunize New Zealand rabbits . LZP3-specific rabbit antiserum was identified by ELISA and Western blot . RESULTS: GST-LZP3 fusion protein was overexpressed and its LZP3-specific antiserum was obtained . CONCLUSION: The successful expression of GST-LZP3 fusion protein in E.coli and the preparation of LZP3-specific rabbit antiserum will be valuable for the study on birth control of Lagurus. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 159 - 62 {Construction and expression of anti-human integrin alphavbeta3 scFv}; Wang C et al.; AIM: To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3 . METHODS: The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene . The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E . coli BL21 (DE3) . RESULTS: SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000 . Western blot confirmed that the protein was labeled with His6 . scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature . The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91% . The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation . CONCLUSION: scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 145 - 7 {Hepatocellular carcinoma associated antigen HCA520, a novel Ca2+-binding protein}; Yang MX et al.; AIM: To analyze the binding activity of the hepatocellular carcinoma associated antigen HCA520 to Ca2+ . METHODS: The HCA520 gene was gained by PCR . Then, it was cloned into the prokaryotic expression vector pGEX-4T-3 . The GST-HCA520 fusion gene was induced to express in E . coli and the expressed product was purified via GST-agarose affinity resin . The fusion protein was confirmed by Western blot analysis . The Ca2+ binding capability of the fusion protein was analyzed by dot blot . RESULTS: The GST-HCA520 fusion gene was constructed and the protein was successfully expressed and purified, which was identified by DNA sequencing and Western blot respectively . The fusion protein could bind to Ca2+ depend on dosages . CONCLUSION: HCA520, a novel hepatocellular carcinoma associated antigen, is a novel Ca2+ binding protein. Clin Microbiol Infect, 2004 Jun, 10(6), 579 - 81 Detection of bacterial DNA in cardiac vegetations by PCR after the completion of antimicrobial treatment for endocarditis; Lang S et al.; PCR with broad-range primers for prokaryotic 16S rRNA genes was used to identify bacterial DNA in tissue from patients undergoing valve replacements following a previous episode of infective endocarditis (IE) . Of eight valves investigated, bacterial DNA was detected in three from patients for whom IE had been treated by antibiotic therapy 5, 12 and 18 months previously . The demonstration of bacterial DNA within resected heart valves suggests either recurrence of infection, treatment failure or the persistence of bacterial debris within the cardiac vegetation . There may also be implications for routine use of PCR in the diagnosis of infection. Biophys J, 2004 Jun, 86(6), 3966 - 80 Detecting rearrangements of shaker and NaChBac in real-time with fluorescence spectroscopy in patch-clamped mammalian cells; Blunck R et al.; Time-resolved fluorescence detection of site-directed probes is a major tool in the investigation of structure-function relationships of voltage-dependent ion channels . However, the technique has been limited so far to the Xenopus-oocyte system making it difficult to study proteins, like, e.g., the prokaryotic sodium channel NaChBac, whose expression in oocytes is insufficient or whose physiological functions are distorted in oocytes . To expand the application of site-directed fluorescence detection to these proteins, we used two techniques-semiconfocal epifluorescence and total internal reflection fluorescence-to detect time-resolved fluorescence changes from site-directed labeled proteins expressed in mammalian cells under patch-clamp conditions, and investigated the characteristics and limitations of the techniques . The voltage-sensitive dye, di-8-ANEPPS, was used to monitor control of the membrane voltage in epifluorescence and total internal reflection fluorescence . Fluorescence changes in patch-clamped cells were recorded from a Shaker channel mutant (M356C) labeled in the S3-S4 linker using semiconfocal epifluorescence . The gating kinetics and fluorescence changes were in accordance with previous studies using fluorescence spectroscopy in Xenopus-oocyte systems . We applied our technique to the prokaryotic sodium channel NaChBac . Voltage-dependent protein-rearrangements of S4 could be detected that are independent of inactivation . Comparison of the S3-S4 linker regions revealed structural differences to the KvAP voltage sensor . The results from the NaChBac channel point to structural requirements for the S3-S4 loop to generate a fluorescence signal. Genes Chromosomes Cancer, 2004 Aug, 40(4), 365 - 70 PRDX4, a member of the peroxiredoxin family, is fused to AML1 (RUNX1) in an acute myeloid leukemia patient with a t(X;21)(p22;q22); Zhang Y et al.; The AML1 gene (also known as RUNX1) at 21q22 codes for core binding factor (CBF) alpha, which forms a heterodimer with CBF beta that acts as a transcriptional activating factor . CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells and is frequently disrupted in leukemia through chromosome translocations . We cloned a novel AML1 partner gene, PRDX4, in an X;21 translocation in a 74-year-old male patient diagnosed with acute myeloid leukemia-M2 . Chromosome analysis detected a t(X;21)(p22;q22) as the sole abnormality in bone marrow samples . The involvement of AML1 was confirmed by fluorescence in situ hybridization studies . Using 3' RACE-PCR, we cloned a fusion between exon 5 of AML1 and exon 2 of PRDX4 . RT-PCR confirmed the fusion and detected another fusion between exon 6 of AML1 and exon 2 of PRDX4, indicating alternative splicing of exon 6 of AML1 in the fusion transcripts . PRDX4 is one of six peroxiredoxin-family genes that are highly conserved in eukaryotes and prokaryotes and are ubiquitously expressed . Peroxiredoxin genes exhibit thioredoxin-dependent peroxidase activity and have been implicated in a number of other cellular functions such as cell proliferation and differentiation . PRDX4 plays a regulatory role in the activation of the transcription factor NF-kappaB and is significantly down-regulated in acute promyelocytic leukemia . This is the first example of antioxidant enzyme involvement in a chromosome translocation in leukemia . Acta Biochim Biophys Sin (Shanghai), 2004 Jun, 36(6), 430 - 6 Human recombinant B7-H3 expressed in E . coli enhances T lymphocyte proliferation and IL-10 secretion in vitro; Zhang GB et al.; To explore the biofunctions of human B7-H3 on activated T lymphocyte, the gene of human B7-H3 encoding the extracellular region (IgV-like and IgC-like domains) was obtained by RT-PCR from human lung cells and subcloned into the prokaryotic expression vector pGEX-5X-3 to express glutathione S-transferase (GST) fusion protein . A 49 kD fusion protein (named as GST/hB7-H3 hereafter) was induced by IPTG and purified by standard methods reported in prokaryotic system . In the presence of the first signal imitated by anti-CD3 monoclonal antibody, T lymphocyte proliferation was observed by incubating purified T cells with soluble GST/hB7-H3 fusion protein by MTT assay . The concentrations of IFN-gamma and IL-10 in the supernatants of T cells were determined by ELISA . The results showed that the GST/hB7-H3 protein produced in bacteria had modest biological activities to proliferate the T lymphocyte and enhance IFN-gamma as well as IL-10 secretion. Methods Mol Biol, 2004, 274, 79 - 92 Purification of plastocyanin and cytochrome c6 from plants, green algae, and cyanobacteria; Navarro JA et al.; Plastocyanin and cytochrome c6 are widely distributed over the oxygen-evolving photosynthetic organisms . The two proteins are functionally equivalent, but strongly differ in their global electrostatic charge . In fact, they are acidic in eukaryotes, but either neutral or basic in cyanobacteria . The difference in their electrostatic features is a critical factor in designing the purification procedure, which must be modified and adapted accordingly . This chapter reports the methods for producing (including cell cultures), isolating and purifying plastocyanin and cytochrome c6-which greatly differ in their isoelectric point-from a number of eukaryotic and prokaryotic organisms. Microbiol Mol Biol Rev, 2004 Jun, 68(2), 301 - 19 Diversity in chemotaxis mechanisms among the bacteria and archaea; Szurmant H et al.; The study of chemotaxis describes the cellular processes that control the movement of organisms toward favorable environments . In bacteria and archaea, motility is controlled by a two-component system involving a histidine kinase that senses the environment and a response regulator, a very common type of signal transduction in prokaryotes . Most insights into the processes involved have come from studies of Escherichia coli over the last three decades . However, in the last 10 years, with the sequencing of many prokaryotic genomes, it has become clear that E . coli represents a streamlined example of bacterial chemotaxis . While general features of excitation remain conserved among bacteria and archaea, specific features, such as adaptational processes and hydrolysis of the intracellular signal CheY-P, are quite diverse . The Bacillus subtilis chemotaxis system is considerably more complex and appears to be similar to the one that existed when the bacteria and archaea separated during evolution, so that understanding this mechanism should provide insight into the variety of mechanisms used today by the broad sweep of chemotactic bacteria and archaea . However, processes even beyond those used in E . coli and B . subtilis have been discovered in other organisms . This review emphasizes those used by B . subtilis and these other organisms but also gives an account of the mechanism in E . coli. Microbiol Mol Biol Rev, 2004 Jun, 68(2), 187 - 206 "Sleeping beauty": quiescence in Saccharomyces cerevisiae; Gray JV et al.; The cells of organisms as diverse as bacteria and humans can enter stable, nonproliferating quiescent states . Quiescent cells of eukaryotic and prokaryotic microorganisms can survive for long periods without nutrients . This alternative state of cells is still poorly understood, yet much benefit is to be gained by understanding it both scientifically and with reference to human health . Here, we review our knowledge of one "model" quiescent cell population, in cultures of yeast grown to stationary phase in rich media . We outline the importance of understanding quiescence, summarize the properties of quiescent yeast cells, and clarify some definitions of the state . We propose that the processes by which a cell enters into, maintains viability in, and exits from quiescence are best viewed as an environmentally triggered cycle: the cell quiescence cycle . We synthesize what is known about the mechanisms by which yeast cells enter into quiescence, including the possible roles of the protein kinase A, TOR, protein kinase C, and Snf1p pathways . We also discuss selected mechanisms by which quiescent cells maintain viability, including metabolism, protein modification, and redox homeostasis . Finally, we outline what is known about the process by which cells exit from quiescence when nutrients again become available. Curr Biol, 2004 May 25, 14(10), 891 - 6 Genome compaction and stability in microsporidian intracellular parasites; Slamovits CH et al.; Microsporidian genomes are extraordinary among eukaryotes for their extreme reduction: although they are similar in form to other eukaryotic genomes, they are typically smaller than many prokaryotic genomes . At the same time, their rates of sequence evolution are among the highest for eukaryotic organisms . To explore the effects of compaction on nuclear genome evolution, we sequenced 685,000 bp of the Antonospora locustae genome (formerly Nosema locustae) and compared its organization with the recently completed genome of the human parasite Encephalitozoon cuniculi . Despite being very distantly related, the genomes of these two microsporidian species have retained an unexpected degree of synteny: 13% of genes are in the same context, and 30% of the genes were separated by a small number of short rearrangements . Microsporidian genomes are, therefore, paradoxically composed of rapidly evolving sequences harbored within a slowly evolving genome, although these two processes are sometimes considered to be coupled . Microsporidian genomes show that eukaryotic genomes (like genes) do not evolve in a clock-like fashion, and genome stability may result from compaction in addition to a lack of recombination, as has been traditionally thought to occur in bacterial and organelle genomes. Mol Microbiol, 2004 Jun, 52(6), 1627 - 39 Visualization of DNA double-strand break repair in live bacteria reveals dynamic recruitment of Bacillus subtilis RecF, RecO and RecN proteins to distinct sites on the nucleoids; Kidane D et al.; We have found that SMC-like RecN protein, RecF and RecO proteins that are involved in DNA recombination play an important role in DNA double-strand break (DSB) repair in Bacillus subtilis . Upon induction of DNA DSBs, RecN, RecO and RecF localized as a discrete focus on the nucleoids in a majority of cells, whereas two or three foci were rarely observed . RecN, RecO and RecF co-localized to the induced foci, with RecN localizing first, while RecO localized later, followed by RecF . Thus, three repair proteins were differentially recruited to distinct sites on the nucleoids, potentially constituting active DSB repair centres (RCs) . RecF did not form regular foci in the absence of RecN and failed to form any foci in recO cells, demonstrating a central role for RecN and RecO in initializing the formation of RCs . RecN/O/F foci were detected in recA, recG or recU mutant cells, indicating that the proteins act upstream of proteins involved in synapsis or post-synapsis . In the absence of exogenous DNA damage, RCs were rare, but they accumulated in recA and recU cells, suggesting that DSBs occur frequently in the absence of RecA or RecU . The results suggest a model in which RecN that forms multimers in solution and high-molecular-weight complexes in cells containing DSBs initiates the formation of RCs that mediate DSB repair with the homologous sister chromosome, which presents a novel concept for DSB repair in prokaryotes. Nucleic Acids Res, 2004 Jun 07, 32(10), 3124 - 35 Print 2004. PeerGAD: a peer-review-based and community-centric web application for viewing and annotating prokaryotic genome sequences; D'Ascenzo MD et al.; PeerGAD is a web-based database-driven application that allows community-wide peer-reviewed annotation of prokaryotic genome sequences . The application was developed to support the annotation of the Pseudomonas syringae pv . tomato strain DC3000 genome sequence and is easily portable to other genome sequence annotation projects . PeerGAD incorporates several innovative design and operation features and accepts annotations pertaining to gene naming, role classification, gene translation and annotation derivation . The annotator tool in PeerGAD is built around a genome browser that offers users the ability to search and navigate the genome sequence . Because the application encourages annotation of the genome sequence directly by researchers and relies on peer review, it circumvents the need for an annotation curator while providing added value to the annotation data . Support for the Gene Ontology vocabulary, a structured and controlled vocabulary used in classification of gene roles, is emphasized throughout the system . Here we present the underlying concepts integral to the functionality of PeerGAD. J Biol Chem, 2004 Aug 13, 279(33), 34890 - 7 Epub 2004 Jun 07. Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities; Liger D et al.; Flavodoxins are involved in a variety of electron transfer reactions that are essential for life . Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins . We describe the 2.0-A resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein . YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer . Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins . YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one . Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp . OY1-2 azoreductase . We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity . We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds. Appl Environ Microbiol, 2004 Jun, 70(6), 3724 - 32 Discovery of the novel candidate phylum "Poribacteria" in marine sponges; Fieseler L et al.; Marine sponges (Porifera) harbor large amounts of commensal microbial communities within the sponge mesohyl . We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid . In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain Bacteria that is moderately related to the Planctomycetes, Verrucomicrobia, and Chlamydia lines of decent . Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named "Poribacteria", to acknowledge the affiliation of the new bacterium with sponges . The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA . Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner . PCR screening with "Poribacteria"-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative . In addition to a report for Planctomycetes, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes. Appl Environ Microbiol, 2004 Jun, 70(6), 3475 - 84 Syntrophic-methanogenic associations along a nutrient gradient in the Florida Everglades; Chauhan A et al.; Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades . It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H . Castro, K . R . Reddy, and A . Ogram, Appl . Environ . Microbiol . 68:6129-6137, 2002) . The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient . Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia . Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs . Most cultivable strains from eutrophic and transition regions clustered within novel lineages . In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones . Novel cultivable Methanosaeta sp . and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions. Appl Environ Microbiol, 2004 Jun, 70(6), 3401 - 6 Evolutionary relationships of primary prokaryotic endosymbionts of whiteflies and their hosts; Thao ML et al.; Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are plant sap-sucking insects that harbor prokaryotic primary endosymbionts (P-endosymbionts) within specialized cells located in their body cavity . Four-kilobase DNA fragments containing 16S-23S ribosomal DNA (rDNA) were amplified from the P-endosymbiont of 24 whiteflies from 22 different species of 2 whitefly subfamilies . In addition, 3-kb DNA fragments containing mitochondrial cytB, nd1, and large-subunit rDNA (LrDNA) were amplified from 17 whitefly species . Comparisons of the P-endosymbiont (16S-23S rDNA) and host (cytB-nd1-LrDNA) phylogenetic trees indicated overall congruence consistent with a single infection of a whitefly ancestor with a bacterium and subsequent cospeciation (cocladogenesis) of the host and the P-endosymbiont . On the basis of both the P-endosymbiont and host trees, the whiteflies could be subdivided into at least five clusters . The major subdivision was between the subfamilies Aleyrodinae and Aleurodicinae . Unlike the P-endosymbionts of may other insects, the P-endosymbionts of whiteflies were related to Pseudomonas and possibly to the P-endosymbionts of psyllids . The lineage consisting of the P-endosymbionts of whiteflies is given the designation "Candidatus Portiera" gen . nov., with a single species, "Candidatus Portiera aleyrodidarum" sp . nov. J Mol Biol, 2004 Jun 25, 340(1), 29 - 37 ADP-binding to origin recognition complex of Saccharomyces cerevisiae; Takenaka H et al.; The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p . Orc1p possesses ATPase activity . As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication . We investigated ADP-binding to ORC by a filter-binding assay . The K(d) values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP . ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay . ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A . Origin DNA fragments did not stimulate ADP-binding to any type of ORC . In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner . Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes . However, overall control may be different . In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods. Clin Immunol, 2004 Jun, 111(3), 286 - 96 Identification of candidate microbial sequences from inflammatory lesion of giant cell arteritis; Gordon LK et al.; Giant cell arteritis (GCA) is a granulomatous inflammatory disease of medium and large arteries which is prevalent in the elderly population . The etiology of GCA is unknown, although the immunologic features suggest the possible presence of a microorganism . Our group has examined whether microbial DNA fragments were present at GCA lesions and whether such microbial fragments could be associated with disease pathogenesis . Initial identification of microbial sequences was performed using genomic representational difference analysis (RDA) . Laser dissecting microscopy was used to isolate cells from GCA lesions and adjacent uninvolved temporal artery . Using genomic RDA, we isolated 10 gene fragments; three of these sequences had high homology with prokaryotic genes and were considered high-priority candidates for further study . An examination of serum from GCA(+) individuals (in contrast to healthy age-matched controls) showed the presence of IgG which recognized in vitro translated proteins from these clones. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 585 - 7 {Construction, expression and functional characterization of disulfide-stabilized anti-hepatocarcinoma single chain Fv fused with truncated Pseudomonas exotoxin}; Zhao J et al.; AIM: To prepare fusion protein of disulfide-stabilized anti-hepatocarcinoma single chain Fv(dsFv)and truncated Pseudomonas exotoxin(PE38) . METHODS: The gene encoding the fusion protein was cloned into the prokaryotic expression vector pTIG which was then transformed into E.coli origami(DE3) . The expressed product was purified through Ni-NTA agarose affinity chromatography column . The cytotoxicity of the purified dsFv-PE38 was detected by MTT colorimetry . RESULTS: The immunotoxin dsFv PE38 was expressed in E.coli origami(DE3) in a soluble form, which accounted for nearly 21% of the total bacterial soluble proteins . The results of cytotoxic assay showed that the dsFv-PE38 could specifically kill hepatocarcinoma cells, while spared the normal hepatic cells . CONCLUSION: The immunotoxin dsFv-PE38 may have some potential value in the treatment of hepatocarcinoma. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 582 - 4 {Cloning, prokaryotic expression of CML66 and preparation of its polyclonal antibody}; You Q et al.; AIM: To clone and express CML66 cDNA and to prepare rabbit anti-CML66 antibody . METHODS: cDNA isolated from the testis using RT-PCR was cloned into pGEMT . After sequencing, the cDNA was inserted into prokaryotic expression vector pET32b(+) . The recombinant vector was transformed into BL-21(DE3) through electroporation . 6xHis-tagged CML66 expression was then induced by IPTG . The protein was purified through Ni(2+) affinity chromatography column and characterized by SDS-PAGE and Western blot . The purified protein was injected into rabbits to prepare polyclonal antibody . RESULTS: The cloned cDNA sequence was identical with that previously reported . The target protein was successfully purified . And rabbit's anti-serum with high titer was obtained . CONCLUSION: We have cloned CML66 successfully, expressed and purified the protein in E.coli.Furthermore,rabbit polyclonal antibody has been obtained. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 570 - 3 {Cloning and expression of Fab gene of mAb HAb18 against human hepatocellular carcinoma}; Xing JL et al.; AIM: To clone Fab gene of mAb HAb18 against human hepatocellular carcinoma and express it in E.coli . METHODS: The cDNAs of kappa chain and Fd of mAb HAb18 were amplified by RT-PCR and cloned into prokaryotic expression vector pComb3 which was transfected into competent E.coli to express Fab by IPTG induction . The specificity of the expressed Fab was tested by ELISA and immunofluorescence staining . RESULTS: The Fab gene of mAb HAb18 was successfully amplified and expressed in E.coli . The results of competitive ELISA and immunofluorescence staining showed that the expressed Fab had specific antigen-binding activity . CONCLUSION: HAb18 Fab was prepared successfully, which lays the foundation for its further application to diagnosis and therapy of human hepatocellular carcinoma. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 535 - 7 {Expression and biological activities of TRAIL fusion protein gene}; Li J et al.; AIM: To obtain and express the full-length of TRAIL gene in prokaryocytes . METHODS: The full-length of TRAIL gene was amplified from peripheral blood cells(PBCs)by RT-PCR and then cloned into the expression vector pGEX-2T . The TRAIL-GST was expressed in E.coli . RESULTS: Sequence analysis indicated that the sequence of TRAIL gene was identical with that in the GenBank . The TRAIL-GST was expressed in E.coli . Expressed product could inhibit the proliferation of KB and Hela cells and finally lead to their apoptosis . CONCLUSION: The TRAIL-GST with biological activity has been expressed in the prokaryotic system. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jan, 20(1), 109 - 12 {Expression, purification and identification of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27)}; Liu YQ et al.; AIM: To construct prokaryotic expression vector of recombinant human lymphotoxin alpha deletant (rhLT-alphaDeltaN27) and express the protein in E.coli . METHODS: The rhLT-alphaDeltaN27 gene was amplified by RT-PCR using total RNA extracted from Jurkat cells,cloned into prokaryotic expression vector pET-23b, and transformed into E.coli BL21(DE3) . The recombinant protein was expressed after IPTG induction and purified by DEAE Sepharose FF and Phenyl-Sepharose FF . RESULTS: The recombinant protein was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein.After purification, the purity of rhLT-alphaDeltaN27 was 99%, and the biological activity was more than 8x10(7) U/mg . Other characteristics of rhLT-alphaDeltaN27, such as relative molecular mass(M(r)), pI and N-terminal amino acid sequence, all corresponded to theoretical prediction . CONCLUSION: The expression vector of rhLT-alphaDeltaN27 gene was constructed, and the recombinant protein was expressed in E.coli successfully.A method of for purifying rhLT-alphaDeltaN27 was established. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jan, 20(1), 91 - 4 {Preparation and characterization of rabbit antibody against human selenoprotein P}; Fang Q et al.; AIM: To prepare rabbit antibody against human selenoprotein P (HSelP) by using HSelP expressed in the prokaryotic expression system and identify its specificity . METHODS: HSelP gene fragment was expressed in E.Coli via IPTG induction and purified through DEAE and Ni-NTA columns sequentially . The purified HSelP as immunogen was used to immunize rabbit . The specificity of rabbit anti-HSelP antibody was analyzed by Western blot . RESULTS: HSelP protein had been expressed and purified successfully . The polyclonal anti-HSelP antibody could specifically recognize natural HSelP from human serum . CONCLUSION: Polyclonal anti-HSelP antibody with high specificity and purity has been prepared by using purified HSelP as immunogen, which lays the foundation for further research on the function and distribution of HSelP, as well as developing a simple method for detecting HSelP. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jan, 20(1), 62 - 6 {Construction, expression and antigenicity of bivalent vaccine candidate of human Helicobacter pylori}; Jiang Z et al.; AIM: To construct a recombinant vector containing fused gene of heat shock protein A(HspA) and outer membrane protein(OMP) with M(r) 18,000, from human Helicobacter pylori(Hp) and express the fusion protein in E.coli BL21 . METHODS: The gene encoding HspA was amplified from Hp chromosome by PCR . After digestion with kpn I and BamH I, the HspA gene was inserted into the prokaryotic expression vector pET32a(+) . After recombinant vectors pET32a(+)/HspA and pET32a(+)/Omp (18) were digested with Hind III and BamH I, the pET32a(+)/HspA and 18,000 OMP gene segments were recovered through agarose electrophoresis, and connected by T4 ligase . The recombinant vector pET32a(+)/HspA-Omp(18) was transformed into E.coli BL21(DE3) . The antigenicity of recombinant fusion protein was analysed by Western blot . RESULTS: Enzyme digestion analysis and sequencing showed that the fused gene had 891 base pairs . As compared with gene reported in GenBank, there were 1.15% and 1.26% differences in cloned nucleotide sequence and amino acid sequence respectively . SDS-PAGE analysis showed that recombinant vector could be expressed in E.coli BL21 . The relative molecule mass (M(r)) of expressed fusion protein was 51x10(3) . And soluble expression product accounted for 18.96% of total bacterial protein.After purification via Ni-NTA agarose resin, the purity of recombinant fusion protein was about 95% . The Western blot analysis showed that recombinant fusion protein could be recognized by anti-Hp positive serum and anti-18,000 OMP mAb, suggesting that this protein had good antigenicity . CONCLUSION: The fused gene of HspA and OMP is cloned and expressed successfully, which lays the foundation for development of protein and DNA vaccines and a diagnostic kit of Hp infection. Immunol Res, 2004, 29(1-3), 175 - 86 Bacterial inhibition of eukaryotic pro-inflammatory pathways; Neish AS; Eukaryotic cells perceive and respond to microbes, both pathogenic and commensal, by activation of signaling cascades such as the NF-kappaB pathway . Induction of such pathways leads to the upregulation of a program of gene expression that mediates pro-inflammatory and anti-apoptotic effector proteins . This host response is usually effective in clearing or controlling an infection . For pathogens (and potentially, symbionts) to continue their lifecycle, it is necessary to evade or repress these cellular responses . There has been recent interest in bacteria that can inhibit pro-inflammatory pathways, in some cases by the actions of soluble effector proteins secreted via a Type III secretion system . Certain effector proteins with this function possess enzymatic activity toward ubiquitin and related molecules . Ubiquitination is an increasingly recognized regulatory modification in eukaryotic cells, and microbial effectors that modulate this key host system may have diverse effects on infected cells and mediate many aspects of eukaryotic-prokaryotic interactions. Curr Drug Targets Infect Disord, 2004 Jun, 4(2), 111 - 6 Antiviral properties of quinolone-based drugs; Richter S et al.; Quinolones represent an important class of broad-spectrum antibacterials, the main structural features of which are a 1,4 dihydro-4-oxo-quinolinyl moiety bearing an essential carboxyl group at position 3 . Quinolones inhibit prokaryotic type II topoisomerases, namely DNA gyrase and, in a few cases, topoisomerase IV, through direct binding to the bacterial chromosome . Based on the hypothesis that these drugs could also bind to the viral nucleic acids or nucleoprotein-complexes, several quinolone derivatives were tested for their antiviral activity . Indeed, antibacterial fluoroquinolones were shown to be effective against vaccinia virus and papovaviruses; these preliminary results prompted the synthesis of modified quinolones to optimize antiviral action and improve selectivity index . The introduction of an aryl group at the piperazine moiety of the fluoroquinolone shifted the activity from antibacterial to antiviral, with a specific action against HIV . The antiviral activity seemed to be related to an inhibitory effect at the transcriptional level, and further evidence suggested a mechanism of action mediated by inhibition of Tat functions . Substitution of the fluorine at position 6 with an amine group to give aryl-piperazinyl-6-amino-quinolones improved the activity and selectivity against HIV-1: the most potent compound of this series was shown to inhibit virus replication through interference with Tat-TAR interaction . A comprehensive SAR investigation was performed based on additional chemical intervention to the quinolone template moiety, such as the introduction of nucleoside derivative functions . The information gained so far will be useful for future rational drug design aimed at developing new compounds with optimized antiviral activity. Protein Expr Purif, 2004 Jul, 36(1), 1 - 10 Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli; You WK et al.; Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis . Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors . In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities . The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC . The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody . The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos . In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model . Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis. Nature, 2004 Jun 3, 429(6991), 558 - 62 Positive selection at sites of multiple amino acid replacements since rat-mouse divergence; Bazykin GA et al.; New alleles become fixed owing to random drift of nearly neutral mutations or to positive selection of substantially advantageous mutations . After decades of debate, the fraction of fixations driven by selection remains uncertain . Within 9,390 genes, we analysed 28,196 codons at which rat and mouse differ from each other at two nucleotide sites and 1,982 codons with three differences . At codons where rat-mouse divergence involved two non-synonymous substitutions, both of them occurred in the same lineage, either rat or mouse, in 64% of cases; however, independent substitutions would occur in the same lineage with a probability of only 50% . All three non-synonymous substitutions occurred in the same lineage for 46% of codons, instead of the 25% expected . Furthermore, comparison of 12 pairs of prokaryotic genomes also shows clumping of multiple non-synonymous substitutions in the same lineage . This pattern cannot be explained by correlated mutation or episodes of relaxed negative selection, but instead indicates that positive selection acts at many sites of rapid, successive amino acid replacement. Mol Cell, 2004 Jun 4, 14(5), 647 - 56 Transcriptional interference between convergent promoters caused by elongation over the promoter; Callen BP et al.; Transcriptional interference with convergent transcription from face-to-face promoters is a potentially important form of gene regulation in all organisms . Using LacZ reporter studies, the mechanism of interference was determined for a pair of face-to-face prokaryotic promoters in which a strong promoter interferes 5.6-fold with a weak promoter, 62 bp away . The promoters were variously rearranged to test different models of interference . Terminating transcription from the strong promoter before it reached the weak promoter dramatically reduced interference, indicating a requirement for the passage of the converging RNAP over the weak promoter . Based on in vitro experiments showing a slow rate of escape for open complexes at the weak promoter and their sensitivity to head-on collisions with elongating RNAP, a "sitting duck" model of interference is proposed and supported with in vivo permanganate footprinting . The model is further supported by the analysis of a second set of prokaryotic face-to-face promoters. Proteomics, 2004 Jun, 4(6), 1562 - 70 The Phosphorylation Site Database: A guide to the serine-, threonine-, and/or tyrosine-phosphorylated proteins in prokaryotic organisms; Wurgler-Murphy SM et al.; The Phosphorylation Site Database {provides ready access to information from the primary scientific literature concerning those proteins from prokaryotic organisms, i.e., the members of the domains Archaea and Bacteria, that have been reported to undergo covalent phosphorylation on the hydroxyl side chains of serine, threonine, and/or tyrosine residues . Where known, the sequence of the site(s) of phosphorylation and the functional consequences of phosphorylation also are included . Active links enable users to quickly access further information concerning the phosphoprotein of interest from PubMed, GenBank, SWISS-PROT, and PIR. Methods Mol Biol, 2004, 284, 209 - 16 Assaying lipid phosphate phosphatase activities; Han GS et al.; Lipid phosphate molecules such as phosphatidate, lysophosphatidate, and diacylglycerol pyro phosphate play roles as signaling molecules in prokaryotic and eukaryotic cells . The cellular processes by which lipid phosphate molecules signal may be attenuated through the action of lipid phosphate phosphatase enzymes . The levels of lipid phosphate phosphatase activities may be used as a marker of signaling events in the cell . In this chapter we describe enzymatic assays that are routinely used to measure the activities of phosphatidate phosphatase, lysophosphatidate phosphatase, and diacylglycerol pyrophosphate phosphatase . These activities are measured by following the release of water-soluble radioactive inorganic phosphate from chloroform-soluble radioactive lipid phosphate substrate following a simple chloroform/methanol/ water phase partition. Plant Physiol, 2004 Jun, 135(2), 859 - 66 Epub 2004 Jun 01. Expression of the Isochrysis C18-delta9 polyunsaturated fatty acid specific elongase component alters Arabidopsis glycerolipid profiles; Fraser TC et al.; A cDNA isolated from the prymnesiophyte micro-alga Isochrysis galbana, designated IgASE1, encodes a fatty acid elongating component that is specific for linoleic acid (C18:2n-6) and alpha-linolenic acid (C18:3n-3) . Constitutive expression of IgASE1 in Arabidopsis resulted in the accumulation of eicosadienoic acid (EDA; C20:2n-6) and eicosatrienoic acid (ETrA; C20:3n-3) in all tissues examined, with no visible effects on plant morphology . Positional analysis of the various lipid classes indicated that these novel fatty acids were largely excluded from the sn-2 position of chloroplast galactolipids and seed triacylglycerol, whereas they were enriched in the same position in phosphatidylcholine . EDA and ETrA are precursors of arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), and docosahexaenoic acid (C22:6n-3) synthesized via the so-called omega6 Delta8 desaturase and omega3 Delta8 desaturase biosynthetic pathways, respectively . The synthesis of significant quantities of EDA and ETrA in a higher plant is therefore a key step in the production of very long chain polyunsaturated fatty acid in oil-seed species . The results are further discussed in terms of prokaryotic and eukaryotic pathways of lipid synthesis in plants. Genome Res, 2004 Jun, 14(6), 1036 - 42 Bacterial genomes as new gene homes: the genealogy of ORFans in E . coli; Daubin V et al.; Differences in gene repertoire among bacterial genomes are usually ascribed to gene loss or to lateral gene transfer from unrelated cellular organisms . However, most bacteria contain large numbers of ORFans, that is, annotated genes that are restricted to a particular genome and that possess no known homologs . The uniqueness of ORFans within a genome has precluded the use of a comparative approach to examine their function and evolution . However, by identifying sequences unique to monophyletic groups at increasing phylogenetic depths, we can make direct comparisons of the characteristics of ORFans of different ages in the Escherichia coli genome, and establish their functional status and evolutionary rates . Relative to the genes ancestral to gamma-Proteobacteria and to those genes distributed sporadically in other prokaryotic species, ORFans in the E . coli lineage are short, A+T rich, and evolve quickly . Moreover, most encode functional proteins . Based on these features, ORFans are not attributable to errors in gene annotation, limitations of current databases, or to failure of methods for detecting homology . Rather, ORFans in the genomes of free-living microorganisms apparently derive from bacteriophage and occasionally become established by assuming roles in key cellular functions . Photochem Photobiol Sci, 2004 Jun, 3(6), 503 - 11 Epub 2004 May 10. Photoresponsive cAMP signal transduction in cyanobacteria; Ohmori M et al.; The molecular mechanism of cAMP-mediated signal transduction from light reception to the physiological response via regulation of gene expression in cyanobacteria is described based on our recent works . Cyanobacteria are known as the organisms that acquired oxygen-evolving, higher plant type photosynthesis . We have found that the cellular cAMP level in the filamentous cyanobacteria Anabaena was oppositely regulated by red and far-red light, i.e., decreasing and increasing, respectively, suggesting that a phytochrome-like red/far-red photoreversible pigment regulates the activity of a certain adenylate cyclase . On the other hand, in the unicellular cyanobacterium Synechocystis cellular cAMP content was increased by blue light irradiation, which led to stimulation of cell motility . The cAMP signaling pathway is known to play an important role in the regulation of various biological activities by altering enzyme activities or controlling gene expression levels in both prokaryotes and eukaryotes . We have isolated genes for adenylate cyclases and cAMP receptor proteins and characterized their molecular properties . Disruption of these genes resulted in the loss of cell motility . It is concluded that the light signal was transmitted by cAMP signal cascade in cyanobacteria. Photochem Photobiol Sci, 2004 Jun, 3(6), 495 - 502 Epub 2004 May 19. Signal transduction during light-quality acclimation in cyanobacteria: a model system for understanding phytochrome-response pathways in prokaryotes; Stowe-Evans EL et al.; The colorful process of complementary chromatic adaptation (CCA), in which cyanobacteria dramatically alter their pigmentation in response to ambient light color changes, has intrigued scientists for more than a century . Over the past four decades, intensive research on the model organism Fremyella diplosiphon has revealed many details of the photobiology and molecular biology of this process, which includes restructuring of these organism's photosynthetic light-harvesting antennae, called phycobilisomes . This restructuring involves changes in transcription of genes encoding phycobilisome components . These genes have been cloned and their patterns of light-responsive expression characterized . In the past ten years, attention has focused on the signal transduction mechanism(s) through which cyanobacteria sense and respond to changes in ambient light color . Genetic approaches led to the isolation of signal transduction components that control light-color responses in F . diplosiphon . Several of these appear to be within a complex phosphorelay that is in part controlled by a photoreceptor called RcaE, the founding member of a large, novel class of prokaryotic photoreceptors with similarity to both plant phytochrome photoreceptors and sensor histidine kinases . The strong foundation of knowledge provided by years of research on CCA makes this a powerful model system for studying signal transduction systems controlled by prokaryotic phytochromes . In this regard, recent results demonstrate that multiple light sensing systems control this organism's responses to changes in light quality and that large numbers of genes are differentially regulated during this process. Biochem J, 2004 Aug 1, 381(Pt 3), 561 - 79 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis; Rider MH et al.; Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis . Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2 . The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes . The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct . Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase . This was confirmed by X-ray crystallography . A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist . This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution . In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers . There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme . In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors . One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way . As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation . In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation. Biochemistry (Mosc), 2004 Apr, 69(4), 435 - 40 DNA polymerase iota-like activity in crude cell extracts of different mouse organs; Gening LV et al.; The recently discovered DNA polymerase iota differs greatly from the numerous eukaryotic and prokaryotic DNA polymerases known previously in its ability to catalyze error-prone DNA synthesis . Using homogeneous preparations of the enzyme, it was shown previously that DNA polymerase iota incorporated preferentially dGMP opposite the thymidine of the template in the growing DNA chain . To elucidate the role of this enzyme in the mammals, its activity was assayed in crude cell extracts of different mouse organs . It is shown that the extracts of the brain and testis cells exhibit the highest activity of DNA polymerase iota, which is not in agreement with the results of other authors . The data suggest that the tissue specific expression of DNA polymerase iota is regulated to a significant degree at the posttranscriptional and posttranslational levels. Biochemistry (Mosc), 2004 Apr, 69(4), 401 - 6 Effects of N-terminal deletion mutation on rabbit muscle lactate dehydrogenase; Zheng Y et al.; Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues . The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b . The mutant enzymes were expressed in E . coli BL21/DE3 and were purified . Then their characteristics and stabilities were studied . The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions . The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers . The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl . The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded . Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH . These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme. Biochemistry, 2004 Jun 8, 43(22), 6917 - 27 Mutagenesis of glutamine 290 in Escherichia coli and mitochondrial elongation factor Tu affects interactions with mitochondrial aminoacyl-tRNAs and GTPase activity; Hunter SE et al.; Elongation factor Tu (EF-Tu) is responsible for the delivery of the aminoacyl-tRNAs (aa-tRNA) to the ribosome during protein synthesis . The primary sequence of domain II of EF-Tu is highly conserved . However, several residues thought to be important for aa-tRNA binding in this domain are not conserved between the mammalian mitochondrial and bacterial factors . One of these residues is located at position 290 (Escherichia coli numbering) . Residue 290 is Gln in most of the prokaryotic factors but is conserved as Leu (L338) in the mammalian mitochondrial factors . This residue is in a loop contacting the switch II region of domain I in the GTP-bound structure . It also helps to form the binding pocket for the 5' end of the aa-tRNA in the ternary complex . In the present work, Leu338 was mutated to Gln (L338Q) in EF-Tu(mt) . The complementary mutation was created at the equivalent position in E . coli EF-Tu (Q290L) . EF-Tu(mt) L338Q functions as effectively as wild-type EF-Tu(mt) in poly(U)-directed polymerization with both prokaryotic and mitochondrial substrates and in ternary complex formation assays with E . coli aa-tRNA . However, the L338Q mitochondrial variant has a reduced affinity for mitochondrial Phe-tRNA(Phe) . E . coli EF-Tu Q290L is more active in poly(U)-directed polymerization with both mitochondrial and prokaryotic substrates and has a higher GTPase activity in both the absence and presence of ribosomes . Surprisingly, while E . coli EF-Tu Q290L is more active in polymerization with mitochondrial Phe-tRNA(Phe), this variant has low activity in the formation of a stable ternary complex with mitochondrial aa-tRNA. J Mol Evol, 2004 May, 58(5), 615 - 31 Detection of lateral gene transfer events in the prokaryotic tRNA synthetases by the ratios of evolutionary distances method; Farahi K et al.; The availability of large numbers of genomic sequences has demonstrated the importance of lateral gene transfer (LGT) in prokaryotic evolution . However, considerable uncertainty remains concerning the frequency of LGT compared to other evolutionary processes . To examine LGTs in ancient lineages of prokaryotes a method was developed that utilizes the ratios of evolutionary distances (RED) to distinguish between alternative evolutionary histories . The advantages of this approach are that the variability inherent in comparing protein sequences is transparent, the direction of LGT and the relative rates of evolution are readily identified, and it is possible to detect other types of evolutionary events . This method was standardized using 35 genes encoding ribosomal proteins that were believed to share a vertical evolution . Using RED-T, an original computer program designed to implement the RED method, the evolution of the genes encoding the 20 aminoacyl-tRNA synthetases was examined . Although LGTs were common in the evolution of the aminoacyl-tRNA synthetases, they were not sufficient to obscure the organismal phylogeny . Moreover, much of the apparent complexity of the gene tree was consistent with the formation of the paralogs in the ancestors to the modern lineages followed by more recent loss of one paralog or the other. Mol Biol Cell, 2004 Aug, 15(8), 3811 - 28 Epub 2004 May 28. Characterization of the Saccharomyces cerevisiae Fol1 protein: starvation for C1 carrier induces pseudohyphal growth; Guldener U et al.; Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines . Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo . We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene . Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS) . All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total . Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy . FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate) . Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth . The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins. J Biol Chem, 2004 Jul 30, 279(31), 32225 - 32 Epub 2004 May 28. Escherichia coli nucleoside diphosphate kinase interactions with T4 phage proteins of deoxyribonucleotide synthesis and possible regulatory functions; Shen R et al.; In both prokaryotic and eukaryotic organisms, nucleoside diphosphate kinase is a multifunctional protein, with well defined functions in ribo- and deoxyribonucleoside triphosphate biosynthesis and more recently described functions in genetic and metabolic regulation, signal transduction, and DNA repair . This paper concerns two unusual properties of nucleoside diphosphate (NDP) kinase from Escherichia coli: 1) its ability to interact specifically with enzymes encoded by the virulent bacteriophage T4 and 2) its roles in regulating metabolism of the host cell . By means of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays, we have shown that E . coli NDP kinase interacts directly with T4 thymidylate synthase, aerobic ribonucleotide reductase, dCTPase-dUTPase, gene 32 single-strand DNA-binding protein, and deoxycytidylate hydroxymethylase . The interactions with ribonucleotide reductase and with gp32 are enhanced by nucleoside triphosphates, suggesting that the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool . The other investigations in this work stem from the unexpected finding that E . coli NDP kinase is dispensable for successful T4 phage infection, and they deal with two observations suggesting that the NDP kinase protein plays a genetic role in regulating metabolism of the host cell: 1) the elevation of CTP synthetase activity in an ndk mutant, in which the structural gene for NDP kinase is disrupted, and 2) the apparent ability of NDP kinase to suppress anaerobic growth in a pyruvate kinase-negative E . coli mutant . Our data indicate that the regulatory roles are metabolic, not genetic, in nature. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Sep, 19(5), 483 - 5 {Prokaryotic expression and bioactivity identification of Fab against human gamma-seminoprotein}; Sun JF et al.; AIM: To construct the expression vector containing cDNA encoding Fab against human gamma-seminoprotein and express it in E . coli . METHODS: The genes encoding K chain and Fd against gamma-seminoprotein were acquired from pUC19-K and pBluescript KS( M13-)-Fd by restrictive enzyme digestion and then cloned into the expression vector pComb3 to construct recombinant expression vector pComb3-Fab . pComb3-Fab was transfected into and expressed in XLI-Blue . RESULTS: Fab against r-semino-protein was expressed in . XLI-Blue . Western blot analysis and immunocytochemical staining demonstrated that ex-pressed Fab could specifically bind to gamma-seminoprotein.CONCLUSION: Fab against gamma -seminoprotein has been expressed successfully with biological activity, which create favourable condition for further study on targeted therapy of prostate cancer. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Sep, 19(5), 480 - 2 {Construction and expression of prokaryotic expression vector of GAP-43 and preparation of monoclonal antibody against GAP-43}; Duan XL et al.; AIM: To express the growth-associated protein-43 (GAP-43)in prokaryotic cells and prepare monoclonal antibody( mAb)against GAP-43 . METHODS: Full length sequence of GAP-43 gene was amplified from the plasmid containing pGAP-43cDNA and was cloned into the expression vector pGEX-4T-I.GST-GAP-43 fusion protein was expressed in E . coil under IPTG induction . Expressed fusion protein was purified by glutathine agarose chromagraphy, Using purified protein as an immunogen, mAb against GAP-43 was prepared . RESULTS: The recombinant plasmid containing the target gene was constructed successfully . The fusion protein was expressed in E . coli in soluble form . The titer of anti-GAP-43mAb in ascites was 1: 10' . The Ig subclass and subtype of the mAb was IgG2a and K type, respectively . Specificity of the mAb was confirmed by ELISA, Western blot and immunofluorescence technique . CONCLUSION: The anti-GAP-43 mAb obtained has strong specificity and high titer, which provides an useful reagent for further studying the function of GAP-43 in nervous system. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Sep, 19(5), 443 - 5 {Cloning and prokaryotic expression of heat shock protein 70 gene of Mycobacterium tuberculosis}; Ye J et al.; AIM: To clone the heat shock protein 70 (Hsp70) gene of Mycobacterium tuberculosis and express it in E.coli . METHODS: Hsp70 gene was amplified by PCR from Mycobacterium tuberculosis H37Rv, then inserted into pUC19 vector and sequenced . The recombinant expression plasmid pGEX-TBhsp70 was constructed and expressed in E.coli . RESULTS: Hsp70 gene was obtained form Mycobacterium tuberculosis and its sequence was identical with that reported in GenBank . The E.coli . containing pGEX-TBhsp70 could express a protein with M(r) being 96,000 after induction of IPTG . CONCLUSION: A confirmed Hsp70 gene is cloned and expressed in E.coli successfully, which lay the foundation for the related research of the gene. Inflamm Bowel Dis, 2004 Mar, 10(2), 159 - 68 Molecular aspects of intestinal epithelial cell-bacterial interactions that determine the development of intestinal inflammation; Neish AS; The mechanisms by which intestinal epithelial cells perceive and respond to microbes, both pathogenic and commensal, is important to understand the pathogenesis of inflammatory diseases . Recent work has established that most eukaryotic cells possess families of receptors that can detect the structural signatures of prokaryotic life . Cells respond to the detection of microbes by activation of distinct cytoplasmic signaling cascades that ultimately result in the transcriptional activation of programs of genes with proinflammatory and anti-apoptotic function . These responses generally suffice to eliminate microbial threats . Also important are potential mechanisms by which microbes can influence the intestinal epithelial responses, influences with significant implications for the normal function of the intestine and inflammatory diseases. Bioinformatics, 2004 Nov 1, 20(16), 2787 - 91 Epub 2004 May 27. BacTregulators: a database of transcriptional regulators in bacteria and archaea; Martinez-Bueno M et al.; MOTIVATION: The BacTregulators database is intended to collect and to integrate information on proteins belonging to defined families of transcriptional regulators in prokaryotes . RESULTS: The BacTregulators database currently contains data on two families of transcriptional regulators: AraC-XylS and TetR . The proteins included in the BacTregulators database have been identified by screening 123 genomes from archaea and bacteria and the SWISS-PROT and TrEMBL databases with profiles defining each family . As the result of an integration process, we have included 1326 different protein sequences from the AraC-XylS family and 1487 different protein sequences from the TetR family . The definition of an entry in BacTregulators is based on protein sequence, source organism, genome element and position in this genome element . The BacTregulators site allows the user to retrieve protein sequences, functional features and experimental evidence supporting the functions, references and the three-dimensional structure of the regulator when available . BacTregulators supplies an innovative tool that allows the researcher to obtain an integrated report that shows the data corresponding to other entries which are related by sequence similarity to the query entry . BacTregulators detects and classifies the regulators belonging to AraC-XylS and TetR families present in prokaryotic genomes, and thus contributes to a more accurate annotation of regulators in genomes . The information collected on each protein in the family can be useful to characterize a new regulator or compile information on the biological properties of a known regulator . AVAILABILITY: The BacTregulators is available at www.bactregulators.org Bioinformatics . 2004 May 27; {Epub ahead of print} Whole-genome prokaryotic phylogeny; Henz SR et al.; Current understanding of the phylogeny of prokaryotes is based on the comparison of the highly conserved small ssu-rRNA subunit and similar regions . Although such molecules have proved to be very useful phylogenetic markers, mutational saturation is a problem, due to their restricted lengths . Now, a growing number of complete prokaryotic genomes are available . This paper addresses the problem of determining a prokaryotic phylogeny utilizing the comparison of complete genomes . We introduce a new strategy, GBDP, "genome blast distance phylogeny", and show that different variants of this approach robustly produce phylogenies that are biologically sound, when applied to 91 prokaryotic genomes . In this approach, first BLAST is used to compare genomes, then a distance matrix is computed, and finally a tree- or network-reconstruction method such as UPGMA, Neighbor-Joining, BioNJ or Neighbor-Net is applied. J Mol Biol, 2004 Jun 11, 339(4), 937 - 49 Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex; de Jager M et al.; Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair . They are grouped based on a conserved set of distinct structural motifs . All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil . Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end . SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins . We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions . We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes . Two distinct architectural arrangements are described, based on the way their components were connected . The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs . These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism. J Mol Biol, 2004 Jun 11, 339(4), 797 - 804 Location of tyrosine 315, a target for phosphorylation by cAbl tyrosine kinase, at the edge of the subunit-subunit interface of the human Rad51 filament; Conilleau S et al.; Rad51 is a key element of recombinational DNA repair and its activity is regulated by phosphorylation of the tyrosine residue at position 315 by cAbl kinase . This phosphorylation could be involved in the resistance of cancer cells to chemotherapy . We have investigated the role of this residue by comparing the three-dimensional structures of human Rad51 and its prokaryotic homologue, Escherichia coli RecA . The residue appeared to be on the edge of the subunit-subunit interacting site . The fluorescence intensity of the tryptophan residue inserted at position 315 of human Rad51 in the place of tyrosine was decreased by adding 3 M urea, although the protein was not unfolded as there was no large change in the fluorescence peak position or circular dichroism signal . This change in fluorescence occurred at a lower urea concentration when the protein was diluted, which favours dissociation . These results indicate that the change is related to the dissociation of Rad51 polymer and that residue 315 is close to the subunit-subunit interacting site . ATP and ADP, which affect the filament structure, caused a blue shift in the fluorescence peak . These nucleotides probably altered the subunit-subunit contacts and may thus affect the filament structure . Phosphorylation of this residue could therefore affect the formation and structure of the Rad51 filament . Correct prediction of subunit-subunit interface of Rad51 by simple comparison of structures of Rad51 and RecA supports the idea that Rad51 forms the filament in a similar way as does RecA. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Jul, 19(4), 409 - 12 {Study on the preliminary purification and bioactivity of recombinant human TNF-alpha mutein 471}; Li YC et al.; AIM: To purify preliminary recombinant human TNF-alpha mutein 471 and detect its bioactivity on the basis of the TNF-alpha mutein 471 expressed in prokaryotic express system . METHODS: The expression of recombinant human TNF-alpha mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression . After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF-alpha mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined . The bioactivities of wild type TNF-alpha and the TNF-alpha mutein 471 were detected by MTT colorimetry . RESULTS: The TNF-alpha mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation . The cytotoxic activity of the TNF-alpha mutein 471 to the L929 cells was 15 times as much as wild type TNF-alpha . CONCLUSION: The TNF-alpha mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Jul, 19(4), 332 - 4 {Construction of the prokaryotic expression vector of MTB lhp gene and its expression}; Chen Q et al.; AIM:To construct prokaryotic expression vector carrying lhp gene and express it in E.coli . METHODS: The MTB lhp gene was amplified by PCR and then cloned into plasmid pQE30 . After sequencing, the gene was cloned into plasmid pET32a(+) to construct recombinant prokaryotic expression vectors pQE30-CFP10 and pET32a(+)-CFP10 . RESULTS: After transformation of the E.coli and induction with 1 mmol/L of IPTG, no additional protein was expressed in pQE30-CFP10 system, but recombinant target protein with M(r) 20 000 or so was expressed in pET32a(+)-CFP10 system, and the expressed protein was maximum when induced with IPTG for 4 h . The expressed protein existed in cytoplasm in soluble form and amounted to 38% of total protein of E.coli . Western blot analysis showed that the protein had good antigenicity . The purity of the protein purified through the Ni-NTA resin reached 93% . CONCLUSION: The prokaryotic expression vector pET32a(+)-CFP10 was constructed successfully and the rCFP10 protein was obtained, which laid the foundation for application of the rCFP10. Brief Funct Genomic Proteomic, 2004 Apr, 3(1), 35 - 46 Yeast as a model organism for studying the evolution of non-standard genetic codes; Silva RM et al.; During the last 30 years, a number of alterations to the standard genetic code have been uncovered both in prokaryotes and eukaryotic nuclear and mitochondrial genomes . But, the study of the evolutionary pathways and molecular mechanisms of codon identity redefinition has been largely ignored due to the assumption that non-standard genetic codes can only evolve through neutral evolutionary mechanisms and that they have no functional significance . The recent discovery of a genetic code change in the genus Candida that evolved through an ambiguous messenger RNA decoding mechanism is bringing that naive assumption to an abrupt end by showing, in a rather dramatic way, that genetic code changes have profound physiological and evolutionary consequences for the species that redefine codon identity . In this paper, the recent data on the evolution of the Candida genetic code are reviewed and an experimental framework based on forced evolution, molecular genetics and comparative and functional genomics methodologies is put forward for the study of non-standard genetic codes and genetic code ambiguity in general . Additionally, the importance of using Saccharomyces cerevisiae as a model organism for elucidating the evolutionary pathway of the Candida and other genetic code changes is emphasised. BMC Bioinformatics . 2004 May 27;5(1):66. Esub8: a novel tool to predict protein subcellular localizations in eukaryotic organisms; Cui Q et al.; BACKGROUND: Subcellular localization of a new protein sequence is very important and fruitful for understanding its function . As the number of new genomes has dramatically increased over recent years, a reliable and efficient system to predict protein subcellular location is urgently needed . RESULTS: Esub8 was developed to predict protein subcellular localizations for eukaryotic proteins based on amino acid composition . In this research, the proteins are classified into the following eight groups: chloroplast, cytoplasm, extracellular, Golgi apparatus, lysosome, mitochondria, nucleus and peroxisome . We know subcellular localization is a typical classification problem; consequently, a one-against-one (1-v-1) multi-class support vector machine was introduced to construct the classifier . Unlike previous methods, ours considers the order information of protein sequences by a different method . Our method is tested in three subcellular localization predictions for prokaryotic proteins and four subcellular localization predictions for eukaryotic proteins on Reinhardt's dataset . The results are then compared to several other methods . The total prediction accuracies of two tests are both 100% by a self-consistency test, and are 92.9% and 84.14% by the jackknife test, respectively . Esub8 also provides excellent results: the total prediction accuracies are 100% by a self-consistency test and 87% by the jackknife test . CONCLUSIONS: Our method represents a different approach for predicting protein subcellular localization and achieved a satisfactory result; furthermore, we believe Esub8 will be a useful tool for predicting protein subcellular localizations in eukaryotic organisms. J Cell Sci, 2004 May 15, 117(Pt 12), 2449 - 60 Mechanosensitive ion channels: molecules of mechanotransduction; Martinac B; Cells respond to a wide variety of mechanical stimuli, ranging from thermal molecular agitation to potentially destructive cell swelling caused by osmotic pressure gradients . The cell membrane presents a major target of the external mechanical forces that act upon a cell, and mechanosensitive (MS) ion channels play a crucial role in the physiology of mechanotransduction . These detect and transduce external mechanical forces into electrical and/or chemical intracellular signals . Recent work has increased our understanding of their gating mechanism, physiological functions and evolutionary origins . In particular, there has been major progress in research on microbial MS channels . Moreover, cloning and sequencing of MS channels from several species has provided insights into their evolution, their physiological functions in prokaryotes and eukaryotes, and their potential roles in the pathology of disease. J Biol Chem, 2004 Jul 30, 279(31), 32474 - 82 Epub 2004 May 24. A homolog of prokaryotic thiol disulfide transporter CcdA is required for the assembly of the cytochrome b6f complex in Arabidopsis chloroplasts; Page ML et al.; The c-type cytochromes are defined by the occurrence of heme covalently linked to the polypeptide via thioether bonds between heme and the cysteine sulfhydryls in the CXXCH motif of apocytochrome . Maintenance of apocytochrome sulfhydryls in a reduced state is a prerequisite for covalent ligation of heme to the CXXCH motif . In bacteria, a thiol disulfide transporter and a thioredoxin are two components in a thio-reduction pathway involved in c-type cytochrome assembly . We have identified in photosynthetic eukaryotes nucleus-encoded homologs of a prokaryotic thiol disulfide transporter, CcdA, which all display an N-terminal extension with respect to their bacterial counterparts . The extension of Arabidopsis CCDA functions as a targeting sequence, suggesting a plastid site of action for CCDA in eukaryotes . Using PhoA and LacZ as topological reporters, we established that Arabidopsis CCDA is a polytopic protein with within-membrane strictly conserved cysteine residues . Insertional mutants in the Arabidopsis CCDA gene were identified, and loss-of-function alleles were shown to impair photosynthesis because of a defect in cytochrome b(6)f accumulation, which we attribute to a block in the maturation of holocytochrome f, whose heme binding domain resides in the thylakoid lumen . We postulate that plastid cytochrome c maturation requires CCDA, thioredoxin HCF164, and other molecules in a membrane-associated trans-thylakoid thiol-reducing pathway. Mol Immunol, 2004 Jun, 41(2-3), 191 - 9 Recombinant cobra venom factor; Vogel CW et al.; Cobra venom factor (CVF) is the complement-activating protein from cobra venom . CVF is a three-chain protein that functionally resembles C3b, the activated form of complement component C3 . Like C3b, CVF forms a C3/C5 convertase with factor B in the presence of factor D and Mg(2+) . Although CVF exhibits functional activity of C3b, it structurally resembles the C3b degradation product C3c, which is not able to form a C3/C5 convertase . CVF has become an important research tool to decomplement laboratory animals in order to study the role of complement in host defense, immune response, and pathogenesis of disease . As the Asian cobras of the Naja species are on the list of endangered species, cobra venom as the source for CVF has become increasingly difficult to obtain . Methods have been developed to recombinantly produce active forms of CVF . This manuscript reviews the production of recombinant pro-CVF using both prokaryotic and eukaryotic expression systems . The recombinant production of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells, stably transfected S2 Drosophila melanogaster cells) generates three forms of pro-CVF: single-chain pro-CVF resembling pro-C3, a two-chain form of pro-CVF resembling C3, and another two-chain form of pro-CVF resembling C3b . All three forms of pro-CVF exhibit functional activity of mature, natural CVF . Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg(2+), forms a bimolecular convertase pro-CVF,Bb that exhibits cleaving activity for both C3 and C5, and depletes the serum complement activity . The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase . Recombinant production of functionally active forms of pro-CVF ensures the availability of an important research reagent for future research involving complement depletion . The experimental systems to recombinantly produce active forms of CVF will also be invaluable for studies to delineate the structure/function relationship of CVF and its differences from C3, and to generate human C3 derivatives with CVF-like function ("humanized CVF") for therapeutic complement depletion. Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 155 - 62 Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system; Faury D et al.; The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane . The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K) . The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme . The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif . In S . lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system . When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system . Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC . Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it . However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor . Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate. Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 145 - 54 Lack of evidence for phosphorylation of Arabidopsis thaliana PII: implications for plastid carbon and nitrogen signaling; Smith CS et al.; The PII signal transduction protein is regulated by covalent modification in most prokaryotic organisms . In enteric bacteria PII is uridylylated on a specific tyrosine residue in the T-loop region, while in certain cyanobacteria it is phosphorylated at the serine residue two positions away from the equivalent modified tyrosine of enteric bacteria . Covalent modification functions primarily to signal cellular nitrogen status in prokaryotes . Here we have examined the phospho-status of Arabidopsis thaliana PII under various growth conditions employing a variety of techniques, including in vivo labeling, phosphospecific antibodies, protein phosphatase treatment, mass spectrometry and protein kinase assays . All results indicate that plant PII is not regulated by phosphorylation . Edman sequencing of immunoprecipitated A . thaliana PII revealed the N-terminal sequences AQISSD and QISSDY, indicating that the mature protein is cleaved from its transit peptide in vivo at the site(s) predicted by ChloroP . Western blot analysis also demonstrated that plant PII protein expression varies little with nutrient regime. Acta Trop, 2004 Jun, 91(1), 69 - 81 Role of proteases in host cell invasion by Toxoplasma gondii and other Apicomplexa; Kim K; The process of invasion by apicomplexan parasites is a carefully coordinated process involving the regulated release of specialized secretory organelles . Several lines of evidence suggest that proteases are critical for the assembly and trafficking of organellar content proteins . Further, invasion is accompanied by cleavage and shedding of secreted proteins as host cell invasion occurs . Recent studies in Toxoplasma gondii and other Apicomplexa have led to the identification of proteases that may mediate these processing events . Among these are subtilases, subtilisin-like serine proteinases that have essential roles in processing of secreted proteins in prokaryotes and eukaryotes . Other studies suggest that cysteine proteinases or rhomboid proteases, a newly described class of serine proteinases, may be important . In addition to providing insights into the invasion process, characterization of invasion proteases may lead to identification of novel targets for antiparasitic chemotherapy. FEMS Microbiol Lett, 2004 Jun 1, 235(1), 95 - 9 An origin for arsenobetaine involving bacterial formation of an arsenic-carbon bond; Ritchie AW et al.; Lysed-cell extract of a Pseudomonas sp . was shown to catalyse bioconversion of dimethylarsinoylacetate to arsenobetaine and dimethylarsinate . Provision of the universal methyl donor S-adenosylmethionine to bioconversion mixtures promoted both the rate and extent of arsenobetaine formation . These findings suggest that in the proposed biosynthesis of arsenobetaine from dimethylarsinoylethanol, oxidation (i.e . the formation of the carboxymethyl group of dimethylarsinoylacetate) would precede the reduction and methylation at the arsenic atom . The presence of enzyme(s) capable of methylating dimethylarsinoylacetate in a bacterial isolate from marine mussel (Mylitus edulis), highlights a possible direct involvement of prokaryotic organisms in the biosynthesis of organoarsenic compounds within marine animals. Antimicrob Agents Chemother, 2004 Jun, 48(6), 1993 - 9 Mercury inactivates transcription and the generalized transcription factor TFB in the archaeon Sulfolobus solfataricus; Dixit V et al.; Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms . Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood . Archaea, like bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus . This study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus . In vivo challenge with mercuric chloride instantaneously blocked cell division, eliciting a cytostatic response at submicromolar concentrations and a cytocidal response at micromolar concentrations . The cytostatic response was accompanied by a 70% reduction in bulk RNA synthesis and elevated rates of degradation of several transcripts, including tfb-1, tfb-2, and lacS . Whole-cell extracts prepared from mercuric chloride-treated cells or from cell extracts treated in vitro failed to support in vitro transcription of 16S rRNAp and lacSp promoters . Extract-mixing experiments with treated and untreated extracts excluded the occurrence of negative-acting factors in the mercury-treated cell extracts . Addition of transcription factor B (TFB), a general transcription factor homolog of eukaryotic TFIIB, to mercury-treated cell extracts restored >50% of in vitro transcription activity . Consistent with this finding, mercuric ion treatment of TFB in vitro inactivated its ability to restore the in vitro transcription activity of TFB-immunodepleted cell extracts . These findings indicate that the toxicity of mercuric ion in S . solfataricus is in part the consequence of transcription inhibition due to TFB-1 inactivation. J Mol Microbiol Biotechnol, 2003, 6(3-4), 206 - 10 Divergent promoter organization may be a preferred structure for gene control in Escherichia coli; Yamada M et al.; The prokaryote possesses intriguing promoter arrangements called divergent promoters, which transcribe adjacent genes in opposite directions . The genome analysis in Escherichia coli revealed the occurrence of a large number of divergent promoters, whose total number was estimated to be about 1.6-fold larger than that of single promoters . Surprisingly, more than 65% divergent promoters transcribe functionally unrelated genes . Transcription from one promoter has been proposed and demonstrated to influence that from the other promoter via change in superhelicity in the divergent promoter region . Therefore, the organism may have acquired divergent promoters as a common structure for ingenious gene controls including transcription coupling . Protein Sci, 2004 Jun, 13(6), 1557 - 65 The structure of Ski8p, a protein regulating mRNA degradation: Implications for WD protein structure; Madrona AY et al.; Ski8p is a 44-kD protein that primarily functions in the regulation of exosome-mediated, 3'--> 5' degradation of damaged mRNA . It does so by forming a complex with two partner proteins, Ski2p and Ski3p, which complete a complex that is capable of recruiting and activating the exosome/Ski7p complex that functions in RNA degradation . Ski8p also functions in meiotic recombination in complex with Spo11 in yeast . It is one of the many hundreds of primarily eukaryotic proteins containing tandem copies of WD repeats (also known as WD40 or beta-transducin repeats), which are short ~40 amino acid motifs, often terminating in a Trp-Asp dipeptide . Genomic analyses have demonstrated that WD repeats are found in 1%-2% of proteins in a typical eukaryote, but are extremely rare in prokaryotes . Almost all structurally characterized WD-repeat proteins are composed of seven such repeats and fold into seven-bladed beta propellers . Ski8p was thought to contain five WD repeats on the basis of primary sequence analysis implying a five-bladed propeller . The 1.9 A crystal structure unexpectedly exhibits a seven-bladed propeller fold with seven structurally authentic WD repeats . Structure-based sequence alignments show additional sequence diversity in the two undetected repeats . This demonstrates that many WD repeats have not yet been identified in sequences and also raises the possibility that the seven-bladed propeller may be the predominant fold for this family of proteins. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Mar, 19(2), 148 - 9 {Cloning of human testicular carcinoma antigen MAGE-E1 gene and its expression in E.coli}; Wang L et al.; AIM: To clone and express the testicular carcinoma antigen MAGE-E1 gene in E.coli . METHODS: The cDNA encoding human MAGE-E1 gene was amplified by RT-PCR from human glioma cell line BT-325, then the MAGE-E1 gene was inserted into plasmid pGEM-T easy . After sequencing, the MAGE-E1 was cloned into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant expression vector pGEX-4T-2-MAGE-E1 which was used to transform E.coli . RESULTS: The expressed product reached the highest level at 5 h after IPTG induction . SDS-PAGE and scanning analysis of gel density indicated that the expressed protein was about M(r) 41 000 and account for 35% of the total bacterial protein . CONCLUSION: The high efficient expression of the MAGE-E1 gene fragment lays the foundation for further preparing antibody against MAGE-E1 protein and the tumor vaccine for glioma immunotherapy. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Mar, 19(2), 140 - 1 {Gene cloning and prokaryotic expression of the PID domain of human DOC-2 molecule}; Liu SJ et al.; AIM: To clone the PID domain of human DOC-2 (nDOC-2) and express it in E.coli DH5alpha . METHODS: The cDNA fragment encoding the PID domain of nDOC-2 was amplified by RT-PCR from normal human ovarian tissue and cloned to pUC19 . The DNA fragment from the pUC19-nDOC-2 digested with BamH I and EcoR I was ligated to the BamH I/EcoR I digested prokaryotic expression vector pGEX-4T-1.The expression of fusion protein was induced with IPTG and the expressed product was identified by SDS-PAGE . RESULTS: (1)The sequencing and endonucleases digestion analysis showed that the fragment of nDOC-2 gene was insert into vectors pUC19 and pGEX-4T-1;(2)SDS-PAGE showed the nDOC-2 gene had been expressed in E.coli DH5alpha . CONCLUSION: The PID domain of nDOC-2 was expressed successfully in prokaryote, which makes preparation for further researching the function of DOC-2 and preparing antibodies to DOC-2 protein. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Mar, 19(2), 118 - 20 {Cloning, prokaryotic expression of LAIR and identification of their immunological reactivities}; Xie X et al.; AIM: To clone and express the leukocyte-associated immunoglobulin-like receptor (LAIR) and to identify the immune reactivity of LAIR-2 to anti-LAIR-1 specific monoclonal antibodies(mAb) . METHODS: Genes encoding LAIR-1 and LAIR-2 were cloned by RT-PCR from human peripheral blood mononuclear cells(PBMC) and two leukemia cell lines Jurkat and HL-60 . The extracellular region gene of LAIR1 and LAIR-2 were inserted into vector pGEX-4T-3 expressing GST fusion protein, expressed on IPTG induction and purified through glutathione-sepharose 4B column . The immunological reactivity of expressed LAIR-2 to anti-LAIR-1 mAb was identified by indirect ELISA . RESULTS: LAIR-1 and LAIR-2 cDNAs had been cloned and expressed . Five new LAIR-1 cDNA isoforms were cloned . Among them, two isoforms from HL-60 included LAIR-1 open reading frames (ORF) and three isoforms from Jurkat were LAIR-1 cDNA segments . The LAIR-1 and LAIR-2 showed different immunological reactivities . CONCLUSION: The transcription, processing after transcription and expression of LAIRs may be related to disparities in individuals and disease status . The difference in immunological reactivity may be involved in their structure. Nucleic Acids Res, 2004 May 18, 32(9), 2768 - 75 Print 2004. A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon; Blaise M et al.; Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain . We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA(Asp) QUC anticodon . This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine . Structural mimicry between the tRNA(Asp) anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved among prokaryotes. J Biol Chem, 2004 Aug 6, 279(32), 33837 - 46 Epub 2004 May 18. Evolutionary links as revealed by the structure of Thermotoga maritima S-adenosylmethionine decarboxylase; Toms AV et al.; S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases . Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme . Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation . The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme . Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T . maritima AdoMetDC despite very limited primary sequence identity . The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis . A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T . maritima and human AdoMetDCs. Proc Natl Acad Sci U S A, 2004 Jun 1, 101(22), 8319 - 24 Epub 2004 May 17. Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose; da Silva Xavier G et al.; Per-Arnt-Sim (PAS) domain-containing kinases are common in prokaryotes, but a mammalian counterpart has only recently been described . Although the PAS domain of the mammalian PAS kinase (PASK) is closely related to the bacterial oxygen sensor FixL, it is unclear whether PASK activity is changed in mammalian cells in response to nutrients and might therefore contribute to signal transduction by these or other stimuli . Here, we show that elevated glucose concentrations rapidly increase PASK activity in pancreatic islet beta cells, an event followed by the accumulation of both PASK mRNA and protein . Demonstrating a physiological role for PASK activation, comicroinjection into clonal beta cells of cDNA encoding wild-type PASK, or PASK protein itself, mimics the induction of preproinsulin promoter activity by high glucose concentrations . Conversely, anti-PASK antibodies block promoter activation by the sugar, and the silencing of PASK expression by RNA interference suppresses the up-regulation by glucose of preproinsulin and pancreatic duodenum homeobox 1 gene expression, without affecting glucose-induced changes in the levels of mRNAs encoding glucokinase or uncoupling protein 2 . We conclude that PASK is an important metabolic sensor in nutrient-sensitive mammalian cells and plays an unexpected role in the regulation of key genes involved in maintaining the differentiated phenotype of pancreatic beta cells. Nucleic Acids Res, 2004 May 17, 32(9), 2707 - 15 Print 2004. A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly; Klostermeier D et al.; In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA . Binding causes a conformational change that is required for subsequent binding events . Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly . The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay. J Mol Biol, 2004 Jun 4, 339(3), 505 - 14 The DNA binding protein H-NS binds to and alters the stability of RNA in vitro and in vivo; Brescia CC et al.; H-NS is an abundant prokaryotic transcription factor that preferentially binds to intrinsically bent DNA . Although H-NS has been shown to reduce the transcription of over 100 genes, evidence suggests that H-NS can also affect the translation of some genes . One such gene, rpoS, specifies a sigma factor, RpoS . The ability of H-NS to bind to the rpoS mRNA and the non-coding RNA regulator, DsrA, was tested . Electrophoretic mobility-shift assays yielded an apparent binding affinity of H-NS binding to curved DNA of approximately 1 microM, whereas binding to rpoS mRNA or DsrA RNA was approximately 3 microM . This RNA binding was not prevented by an excess of competitor yeast RNA, suggesting that H-NS specifically bound these RNAs . Footprint analysis with a single strand-specific ribonuclease was used to identify the H-NS binding site(s) on DsrA and rpoS mRNA . Surprisingly, H-NS appeared to enhance the cleavage of DsrA and rpoS mRNA . The enhanced cleavage was at sites that were predicted to be single-stranded and did not result from contaminating nucleases in the H-NS protein preparation or non-specific effects of the nuclease . Quantitative RT-PCR of RNA isolated from wild-type and hns- strains revealed that H-NS also affects the stability of DsrA in vivo . Thus H-NS appears to modulate RNA stability in vivo and in vitro. Biochemistry, 2004 May 25, 43(20), 5930 - 6 Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome; Mulder FA et al.; During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states . Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation . Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome . Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment . The overall structure can be described as three ellipsoids joined by flexible linkers . No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution . In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies . The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12 . These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors. Bioinformatics, 2004 Nov 1, 20(16), 2644 - 55 Epub 2004 May 14. Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway; van Bakel H et al.; MOTIVATION: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes . However, the degree to which these genes share their functional context as members of the same pathway in the prokaryotic domain is less clear . RESULTS: The co-occurrence of gene families involved in Atx1p-mediated copper transport in the genomes and operon structures of 80 non-redundant prokaryotes was investigated . For this purpose, we developed a Web tool (SHOPS) to display the operon context for a given set of proteins . In total, a set of 43 putative operons was identified . These were found to be involved in a variety of pathways and indicate a large diversity in the functional context of the individual gene family members . AVAILABILITY: The SHOPS tool can be found at supplemental data are available at http://humgen.med.uu.nl/publications/vanbakel/pathway/ Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Apr, 21(2), 251 - 4 {Expression and purification of mouse B lymphocyte chemoattractant}; Jiang Y et al.; A DNA fragment encoding mouse B Lymphocyte Chemoattractant (BLC, MV10Kda) was obtained by PCR . The amplified fragment was inserted into prokaryotic expression vector PQE30 . Recombinant protein was expressed in E . Coli XL-1 blue and purified by affinity chromatography on a nickel-nitrilotriacetic acid gel matrix . Then it was identified by sequence analysis and Western blot analysis . The fragment inserted into prokaryotic expression vector PQE30 was identified to be BLC gene fragment by sequence analysis . And a specfic band was shown by Western blot analysis . These findings provide the evidence that the recombinant protein obtained and purified in this study using gene engineering method is mouse B Lymphocyte Chemoattractant. Ai Zheng, 2004 May, 23(5), 602 - 4 {Mycoplasma infection and cancer}; Ning JY et al.; Mycoplasmas are widespread in nature as conditional pathogen, which may be the unique prokaryote that can cohabit with eukaryote and interact permanently with mammalian cells . Mycoplasma infection can be detected in many tumor tissues, continuous infection of mycoplasma can lead to transformation of mammalian cells, up-regulating expression of oncogenes, and some biologic changes of tumor cells, suggesting association of mycoplasma infection with tumorigenesis. Annu Rev Biophys Biomol Struct, 2004, 33, 177 - 98 Molecules of the bacterial cytoskeleton; Lowe J et al.; The structural elucidation of clear but distant homologs of actin and tubulin in bacteria and GFP labeling of these proteins promises to reinvigorate the field of prokaryotic cell biology . FtsZ (the tubulin homolog) and MreB/ParM (the actin homologs) are indispensable for cellular tasks that require the cell to accurately position molecules, similar to the function of the eukaryotic cytoskeleton . FtsZ is the organizing molecule of bacterial cell division and forms a filamentous ring around the middle of the cell . Many molecules, including MinCDE, SulA, ZipA, and FtsA, assist with this process directly . Recently, genes much more similar to tubulin than to FtsZ have been identified in Verrucomicrobia . MreB forms helices underneath the inner membrane and probably defines the shape of the cell by positioning transmembrane and periplasmic cell wall-synthesizing enzymes . Currently, no interacting proteins are known for MreB and its relatives that help these proteins polymerize or depolymerize at certain times and places inside the cell . It is anticipated that MreB-interacting proteins exist in analogy to the large number of actin binding proteins in eukaryotes . ParM (a plasmid-borne actin homolog) is directly involved in pushing certain single-copy plasmids to the opposite poles by ParR/parC-assisted polymerization into double-helical filaments, much like the filaments formed by actin, F-actin . Mollicutes seem to have developed special systems for cell shape determination and motility, such as the fibril protein in Spiroplasma. J Mol Recognit, 2004 May-Jun, 17(3), 186 - 93 On-chip single-cell-based microcultivation method for analysis of genetic information and epigenetic correlation of cells; Yasuda K; We have developed an on-chip single-cell based microcultivation method for analyzing the variability of genetic information stored in single cells and their epigenetic correlations . The method uses four systems: an on-chip cell sorter for purifying the cells in a non-destructive manner; an on-chip single-cell cultivation chip for isolating single cells; an on-chip agarose microchamber system for constructive cell-cell network formation during cultivation; and an on-chip single-cell-based expression analysis system . Using these systems, we could measure the variability of prokaryotic cells and eukaryotic cells having the same DNA and found that, although prokaryotic cells have a large variability in their interdivision times, sister eukaryotic cells having the same DNA synchronized well . We also measured the dynamics of synchronization of beating cardiac myocytes and found that two isolated cells synchronize by one cell following the other after a short pause in beating . These results showed the potential of the on-chip microcultivation method's constructive approach to analyzing cell systems . J Mol Biol, 2004 May 28, 339(2), 447 - 58 Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation; Gelinas AD et al.; DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP . GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range . GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE . The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis . Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP . ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range . Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M) . This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains . Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions . Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction . GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions. FEMS Microbiol Lett, 2004 May 15, 234(2), 215 - 23 Two FHA domains on an ABC transporter, Rv1747, mediate its phosphorylation by PknF, a Ser/Thr protein kinase from Mycobacterium tuberculosis; Molle V et al.; Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role . The present study concerns one of these enzymes, the serine/threonine protein kinase PknF from Mycobacterium tuberculosis . It is shown that, in addition to its autokinase activity, PknF is able to phosphorylate Rv1747, a newly described ABC transporter . This reaction appears to involve two FHA domains of Rv1747 . It is suggested that recruitment and phosphorylation of Rv1747 depen