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Biomedical subjects

L F Wu

Publications and source records attributed to L F Wu.

At least 19 recordsLinked to original sources
PubMed2001

Involvement of the twin-arginine translocation system in protein secretion via the type II pathway.

The general secretory pathway (GSP) is a two-step process for the secretion of proteins by Gram-negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin-arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.

Amino Acid Sequence↗
PubMed2001

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Algorithms↗
PubMed2001

Effect of O2, H2 and redox potential on the activity and synthesis of hydrogenase 2 in Escherichia coli.

The aim of this work was to study the influence of O2 with special emphasis on low oxygen tension, the effect of H2 under various conditions of oxygen tension and the influence of the redox potential in the growth medium on hydrogenase 2 of Escherichia coli. The hydrogenase activity and the content of the large (HybC) and small (HybO) subunits of hydrogenase 2 were compared during turbidostat cultivation in a wild strain and mutant HDK103 lacking hydrogenases 1 and 3. No hydrogenase 2 activity in the mutant HDK103 was observed under aerobic conditions, but it was maximal under anaerobic conditions and half-maximal at an oxygen tension of approximately 4 mbar as is common for enzymes of anaerobic respiration. The content of hydrogenase 2 in both the strains was maximal under anaerobic conditions. In the wild strain, H2 addition enhanced hydrogenase activity and the HybO content under microaerobic conditions only. Under anaerobic conditions endogenous H2 production hindered this effect. Under aerobic conditions, the 02-related negative effect seemed to dominate over the H2-related positive effect. By contrast, in the mutant HDK103, hydrogen influenced neither hydrogenase 2 activity nor its content. A possible role of hydrogenase I in the response of hydrogenase 2 to hydrogen is discussed. Under conditions of different O2 tension, hydrogenase activity in both strains correlated inversely with the value of the redox potential of the medium. The presence of H2 changed this dependence. Thus, the value of the redox potential itself is not a controlling factor for hydrogenase 2.

Aerobiosis↗
PubMed2000

Translocation of jellyfish green fluorescent protein via the Tat system of Escherichia coli and change of its periplasmic localization in response to osmotic up-shock.

The bacterial twin arginine translocation (Tat) pathway is capable of exporting cofactor-containing enzymes into the periplasm. To assess the capacity of the Tat pathway to export heterologous proteins and to gain information about the property of the periplasm, we fused the twin arginine signal peptide of the trimethylamine N-oxide reductase to the jellyfish green fluorescent protein (GFP). Unlike the Sec pathway, the Tat system successfully exported correctly folded GFP into the periplasm of Escherichia coli. Interestingly, GFP appeared as a halo in most cells and occasionally showed a polar localization in wild type strains. When subjected to a mild osmotic up-shock, GFP relocalized very quickly at the two poles of the cells. The conversion from the halo structure to a periplasmic gathering at particular locations was also observed with spherical cells of the DeltarodA-pbpA mutant or of the wild type strain treated with lysozyme. Therefore, the periplasm is not a uniform compartment and the polarization of GFP is unlikely to be caused by simple invagination of the cytoplasmic membrane at the poles. Moreover, the polar gathering of GFP is reversible; the reversion was accelerated by glucose and inhibited by azide and carbonyl cyanide m-chlorophenylhydrazone, indicating an active adaptation of the bacteria to the osmolarity in the medium. These results strongly suggest a relocalization of periplasmic substances in response to environmental changes. The polar area might be the preferential zone where bacteria sense the change in the environment.

Animals↗
PubMed2000

Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli.

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.

Bacterial Proteins↗
PubMed2000

Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor.

Besides SecA and SecB, Escherichia coli cells possess a signal recognition particle (SRP) to target exported proteins to the SecY translocon. Using chemical and site-specific cross-linking in vitro, we show that SRP recognizes the first signal anchor sequence of a polytopic membrane protein (MtlA) resulting in cotranslational targeting of MtlA to SecY and phospholipids of the plasma membrane. In contrast, a possible interaction of SRP with the secretory protein pOmpA is prevented by the association of trigger factor with nascent pOmpA. Trigger factor also prevents SecA from binding to the first 125 amino acids of pOmpA when they are still associated with the ribosome. Under no experimental conditions was SecA found to interact with MtlA. Likewise, virtually no binding of trigger factor to ribosome-bound MtlA occurs even in the complete absence of SRP. Collectively, our results indicate that at the stage of nascent polypeptides, polytopic membrane proteins are selected by SRP for co-translational membrane targeting, whereas secretory proteins are directed into the SecA/SecB-mediated post-translational targeting pathway by means of their preferential recognition by trigger factor.

Adenosine Triphosphatases↗
PubMed2000

Bacterial twin-arginine signal peptide-dependent protein translocation pathway: evolution and mechanism.

The recently identified bacterial Tat pathway is capable of exporting proteins with a peculiar twin-arginine signal peptide in folded conformation independently of the Sec machinery. It is structurally and mechanistically similar to the delta pH-dependent pathway used for importing chloroplast proteins into the thylakoid. The tat genes are not ubiquitously present and are absent from half of the completely sequenced bacterial genomes. The presence of the tat genes seems to correlate with genome size and with the presence of important enzymes with a twin-arginine signal peptide. A minimal Tat system requires a copy of tatA and a copy of tatC. The composition and gene order of a tat locus are generally conserved within the same taxonomy group but vary considerably to other groups, which would exclude an acquisition of the Tat system by recent horizontal gene transfer. The tat genes are also found in the genomes of chloroplasts and plant mitochondria but are absent from animal mitochondrial genomes. The topology of evolution trees suggests a bacterial origin of the Tat system. In general, the twin-arginine signal peptide is capable of targeting any passenger protein to the Tat pathway. However, a structural signal carried by the mature part of a passenger protein can override targeting information in a signal peptide under certain circumstances. Tat systems show a substrate-Tat component specificity and a species specificity. The pore size of the Tat channel is estimated as being between 5 and 9 nm. Operational models of the Tat system are proposed.

Arginine↗
PubMed2000

[Establishment of a method for GUS gene transferring into wheat (Triticum astivum L.) embryos by low energy ion beam implantation].

Physical parameters influencing transformation of wheat mediated by low energy ion beam, including type of ion, parameters of ion energy, dose and dose rate, were studied. Ar+ was regarded as suitable ions implanted in transformation. 20-25 keV of energy, 4.68 x 10(16) ions/cm2 of dose, 2.6 x 10(15) ions/cm2 of dose rate were chosen as appropriate implantation parameters. The suitable culture conditions for induction and growth of callus and the optimal selection scheme were established, After implantation and selection, resistant calli and hygromycin-resistant plantlets were obtained from three varieties. Molecular analysis data proved that GUS gene had integrated into the wheat genome. The mature embryo transformation efficiency of wherat variety Yangmai 5, Yangmai 158, Wanmai 32 reached 9.5%, 10.8%, 11.2% measured in produced hygromycin-resistant callus and 1.4%, 3.4%, 1.7% measured in regenerated plants, respectively, This experiment provides a basis for further investigation of wheat transformation system. Low energy ion beam mediated transformation can be extended to other plant recalcitrant to Agrobacterium tumefaciens as soon as methodological parameters are optimized.

Gene Transfer Techniques↗
PubMed1999

Requirement for phospholipids of the translocation of the trimethylamine N-oxide reductase through the Tat pathway in Escherichia coli.

Trimethylamine N-oxide reductase (TorA) is an anaerobically synthesized molybdoenzyme. It is translocated across the cytoplasmic membrane in a folded conformation via the Tat pathway of Escherichia coli. The requirement for phospholipids for the export of this enzyme was analyzed in the pgsA and pss mutants lacking anionic phospholipids and phosphatidylethanolamine, respectively. Anaerobic growth did not influence phospholipid composition of the pgsA and pss mutants. Interestingly, both pgsA and pss mutations severely retarded the translocation of TorA into the periplasm. Therefore, translocation of proteins through the Tat pathway is dependent on the anionic phospholipids and on lipid polymorphism.

Biological Transport↗
PubMed1999

Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.

Biological Transport↗
PubMed1999

Enzymatic and physiological properties of the tungsten-substituted molybdenum TMAO reductase from Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of +130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto-TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto-substituted TMAO reductase (W-TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo-TMAO reductase (Mo-TorA). W-TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo-TorA. Most importantly, the W-TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.

Dose-Response Relationship, Drug↗
PubMed1999

Isolation and characterization of the nikR gene encoding a nickel-responsive regulator in Escherichia coli.

Expression of the nickel-specific transport system encoded by the Escherichia coli nikABCDE operon is repressed by a high concentration of nickel. By using random transposon Tn10 insertion, we isolated mutants in which expression of the nik operon became constitutive with respect to nickel. We have identified the corresponding nikR gene which encodes a nickel-responsive regulator. Expression of nikR was partially controlled by Fnr through transcription from the nikA promoter region. In addition, a specific transcription start site for the constitutive expression of nikR was found 51 bp upstream of the nikR gene.

Bacterial Proteins↗
PubMed1998

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.

Bacterial Proteins↗
PubMed1997

[Clinical report of 60 cases of diabetic cardio-vascular autonomous neuropathy by stasis removing treatment of combined traditional and Western medicine].

OBJECTIVE: To study the treatment of diabetic cardio-vascular autonomous neuropathy (DCVAN) by TCM-WM following the principle of promoting blood circulation to remove blood stasis. METHODS: Sixty patients with DCVAN were randomly divided into two groups, the treatment group (30 patients) were treated with combined TCM-WM therapy. Based on the control of blood sugar by WM effectively, then use TCM-Tangxinshen ([symbol: see text], TXS) with the function of supplementing the Qi and nourishing Yin, promoting blood circulation to remove blood stasis. Thirty cases of the control group were treated with WM alone. The course of treatment was three months. RESULTS: The total effective rate of the treatment group and the control group was 77.3% and 33.4% respectively, which were significantly different statistically (P < 0.005). CONCLUSION: The therapeutic effect of TCM-WM was better than that of WM.

Adult↗
PubMed1996

Requirement for nickel of the transmembrane translocation of NiFe-hydrogenase 2 in Escherichia coli.

The cellular location of membrane-bound NiFe-hydrogenase 2 (HYD2) from Escherichia coli was studied by immunoblot analysis and by activity staining. Treatment of spheroplasts with trypsin was able to release active HYD2 into the soluble fraction, indicating that HYD2 is attached to the periplasmic side of the cytoplasmic membrane and that HYD2 undergoes a trans-membrane translocation during its biosynthesis. By using a nik mutant deficient in the high affinity specific nickel transport system, we show that the intracellular availability of nickel is essential for the processing of the large subunit and for the transmembrane translocation of HYD2. We also demonstrate that the processing of the precursor, which is related with nickel incorporation, can occur in the membrane-depleted soluble fraction and that it is associated with the increase in resistance to proteolysis of the processed form of the large subunit. The mechanism of the transmembrane translocation of HYD2 is discussed.

Amino Acid Sequence↗
PubMed1996

Involvement of the GroE chaperonins in the nickel-dependent anaerobic biosynthesis of NiFe-hydrogenases of Escherichia coli.

We analyzed the involvement of chaperonins GroES and GroEL in the biosynthesis of the three hydrogenase isoenzymes, HYD1, HYD2, and HYD3, of Escherichia coli. These hydrogenases are NiFe-containing, membrane-bound enzymes composed of small and large subunits, each of which is proteolytically processed during biosynthesis. Total hydrogenase activity was found to be reduced by up to 60% in groES and groEL thermosensitive mutant strains. This effect was specific because it was not seen for another oligomeric, membrane-bound metalloenzyme, i.e., nitrate reductase. Analyses of the single hydrogenase isoenzymes revealed that a temperature shift during the growth of groE mutants led to an absence of HYD1 activity and to an accumulation of the precursor of the large subunit of HYD3, whereas only marginal effects on the processing of HYD2 and its activity were observed under these conditions. A decrease in total hydrogenase activity, together with accumulation of the precursors of the large subunits of HYD2 and HYD3, was also found to occur in a nickel uptake mutant (nik). The phenotype of this nik mutant was suppressed by supplementation of the growth medium with nickel ions. On the contrary, Ni2+ no longer restored hydrogenase activity and processing of the large subunit of HYD3 when the nik and groE mutations were combined in one strain. This finding suggests the involvement of these chaperonins in the biosynthesis of a functional HYD3 isoenzyme via the incorporation of nickel. In agreement with these in vivo results, we demonstrated a specific binding of GroEL to the precursor of the large subunit of HYD3 in vitro. Collectively, our results are consistent with chaperonin-dependent incorporation of nickel into the precursor of the large subunit of HYD3 as a prerequisite of its proteolytic processing and the acquisition of enzymatic activity.

Amino Acid Sequence↗
PubMed1996

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA.

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA gene-encoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.

Amino Acid Sequence↗