Biomedical subjects

Jianguo Xu

Publications and source records attributed to Jianguo Xu.

At least 55 records · Page 3Linked to original sources

PubMed2003

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2.

Disabled-2 (Dab2; also known as p96 and DOC-2) is a signal transduction protein that has been implicated in the control of cell growth. Dab2 is known to be a phosphoprotein, but little is known about the kinases that phosphorylate Dab2. We have found that Dab2 phosphorylation is markedly increased during the mitosis phase of the cell cycle. This phosphorylation is blocked by roscovitine, a selective inhibitor of cyclin-dependent kinases. Dab2 robustly coimmunoprecipitates from cells with the cyclin-dependent kinase cdc2, and purified cdc2 can phosphorylate purified Dab2 fusion proteins in vitro on multiple sites. Cellular phosphorylation of Dab2 by cdc2 promotes the association of Dab2 with Pin1, a peptidylprolyl isomerase that regulates the rate of Dab2 dephosphorylation. These findings reveal that Dab2 is differentially phosphorylated during the cell cycle by cdc2 and provide a potential feedback mechanism by which Dab2 inhibition of cell growth and proliferation may be regulated.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Heterodimerization of alpha 2A- and beta 1-adrenergic receptors.

beta- and alpha(2)-adrenergic receptors are known to exhibit substantial cross-talk and mutual regulation in tissues where they are expressed together. We have found that the beta(1)-adrenergic receptor (beta(1)AR) and alpha(2A)-adrenergic receptor (alpha(2A)AR) heterodimerize when coexpressed in cells. Immunoprecipitation studies with differentially tagged beta(1)AR and alpha(2A)AR expressed in HEK-293 cells revealed robust co-immunoprecipitation of the two receptors. Moreover, agonist stimulation of alpha(2A)AR was found to induce substantial internalization of coexpressed beta(1)AR, providing further evidence for a physical association between the two receptors in a cellular environment. Ligand binding assays examining displacement of [(3)H]dihydroalprenolol binding to the beta(1)AR by various ligands revealed that beta(1)AR pharmacological properties were significantly altered when the receptor was coexpressed with alpha(2A)AR. Finally, beta(1)AR/alpha(2A)AR heterodimerization was found to be markedly enhanced by a beta(1)AR point mutation (N15A) that blocks N-linked glycosylation of the beta(1)AR as well as by point mutations (N10A/N14A) that block N-linked glycosylation of the alpha(2A)AR. These data reveal an interaction between beta(1)AR and alpha(2A)AR that is regulated by glycosylation and that may play a key role in cross-talk and mutual regulation between these receptors.

Cells, Cultured↗

PubMed2003

Modulation of digoxin transport across Caco-2 cell monolayers by citrus fruit juices: lime, lemon, grapefruit, and pummelo.

PURPOSE: To evaluate the effects of fresh lime, lemon, grapefruit, and pummelo juices on the transport of digoxin, a P-glycoprotein (P-gp) substrate, in Caco-2 cell monolayers. METHODS: Bidirectional [3H]-digoxin fluxes across confluent Caco-2 cell monolayers were determined in 0-50% fruit juices at pH 7.4. Verapamil HCl (100 microM) served as positive control. Juice toxicity was evaluated by the 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. RESULTS: Apical-to-basal (A-to-B) digoxin flux was enhanced by 50% fruit juice in the order of lemon > lime > pummelo > grapefruit. The four fruit juices could be divided into two groups based on their effects on transepithelial electrical resistance (TEER), viability, and digoxin transport activity of the Caco-2 cells. Grapefruit and pummelo juices produced similar digoxin transport profiles that were characteristic of those observed with P-gp inhibitors. Both juices decreased net digoxin efflux by 1.2 U per 10% increase in juice concentration and had a propensity to increase cellular TEER at high concentrations (>30%). However, cellular TEER and viability decreased with increasing concentration of lime and lemon juices. Both juices also produced similar digoxin transport profiles, the A-to-B and B-to-A digoxin Papp increasing with increasing juice concentration above 5%. Net digoxin efflux was 30% of control value and relatively independent of juice concentration. These results paralleled the groupings of the four fruits according to their prominent flavonoid pattern and taxonomy. CONCLUSION: The effects of lime, lemon, grapefruit, and pummelo juices on the TEER, viability, and digoxin transport activity of the Caco-2 cells appeared to be dependent on the dominant flavonoid pattern and taxonomy of the citrus fruits.

Beverages↗

PubMed2003

Isolation of Escherichia coli O157:H7 from dung beetles Catharsius molossus.

In an epidemiological survey, Escherichia coli O157:H7 was isolated from the intestine 4 of 113 dung beetle Catharsius molossus captured below ground at Tongshan County, Jiangsu Province of China. In parallel, 10 strains of E. coli O157:H7 were isolated from fecal samples of 383 diarrhea patients from the same region. Most importantly, using pulsed field gel electrophoresis (PFGE) of chromosomal DNA restriction fragments and PCR method, we found that the PFGE pattern and virulence genes of beetle isolates were identical to those of the human isolates, such as Shiga-toxins (stx) and enterohemorrhagic Escherichia coli hemolysin A (EHEC-hlyA). Therefore, dung beetle might acquire pathogenic E. coli O157:H7 through contact with feces of domestic animals.

Animals↗

PubMed2003

Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the Delta tatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB(1) genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA(1) and reduced verotoxicity were detected in the Delta tatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.

Animals↗

PubMed2003

[The effect of TWP on the reactivity of AChR on the end-plate membrane of frog sartorius].

OBJECTIVE: To study the effect of Tripterygium Wilfordii Polyglycosidium (TWP) on the reactivity of n-Acetylcholine receptor (nAChR) on the end plate membrane. METHODS: Using the microionophoretic method, we applied the exogenous acetylcholine chloride (AChCl) to the superficial end-plates of the isolated nonuniform stretched frog sartorius, and acetylcholine potential (AChP) recorded was taken as an indicator of the reactivity of the nAChR in the end plate. RESULTS: 1. After the addition of TWP, the apparent change of AChP was recorded at 20 min in the 20 mg/L group, and at 10 min in the 40 mg/L and 60 mg/L groups respectively. 2. Within the 20 mg/L and 40 mg/L TWP groups, the change in amplitude of AChP showed significant difference with the time (P < 0.05), the greatest being at 60 min. 3. In the 60 mg/L TWP group, action potential was recorded at 40 min, 60 min and 80 min. 4. There was significant difference in the percentage of AChP amplitude change between the three concentration groups (P < 0.05), and the highest percentage of change appeared in the 60 mg/L group. CONCLUSION: TWP can increase the reactivity of nAChR in the end plate membrane, and there exist an apparent concentration-effect relationship and a certain time-effect relationship.

Animals↗

PubMed2003

[Effects of TWP on capacity of muscle contraction].

OBJECTIVE: To investigate the direct effects of Tripterygium Wilfordii Polyglycosidium (TWP) on capability of muscle contraction. METHODS: Using electronic stimulator to stimulate the phrenic nerve of the isolated phrenic nerve diaphragm preparation of 30 rats or directly stimulate the isolated gastrocnemius muscle preparation of 45 toads in vitro, we studied the effects of TWP on capability of muscle intrinsic contraction. RESULTS: TWP in 20 mg/L increased the amplitude of muscle contraction in initial 60 min but did not make further increase of the amplitude of muscle contraction from 60 min to 90 min in the isolated phrenic nerve diaphragm preparation of rat under one-third optimal stimulus. TWP in 40 mg/L and in 60 mg/L did not cause decrease of amplitude of muscle contraction in initial 60 min in isolated phrenic nerve diaphragm preparation of rat under one-third optimal stimulus. TWP in 60 mg/L did not cause decrease of tension of signal-contraction in initial 30 min in isolated gastrocnemius muscle preparation of toad under one-third optimal direct stimulus. Solvent DMSO could obviously reduce the tension of muscle contraction both in isolated phrenic nerve diaphragm preparation of rat and in isolated gastrocnemius muscle preparation of toad under one-third optimal stimulus. CONCLUSION: TWP can limitedly enhance the capability of muscle contraction; Solvent DMSO can restrain muscle contraction.

Animals↗

PubMed2003

Hydroxyethyl starch inhibits NF-kappaB activation and prevents the expression of inflammatory mediators in endotoxic rats.

Hydroxyethyl starch (HES) has been shown to be beneficial in several inflammatory situations, but the mechanisms are unclear. The present study tested the hypothesis that HES has effects on nuclear factor kappa B (NF-kappaB) activation and the expression of inflammatory mediators induced by lipopolysaccharide. Sepsis was induced in male Wistar rats by injection of lipopolysaccharide (LPS, 6 mg/kg, i.p.). At 1 min after the LPS challenge, HES was infused via the right external jugular vein at the following doses: 3.75, 7.5, 15, or 30 ml/kg. NF-kappaB activation in peripheral blood mononuclear cells and neutrophils, plasma concentrations of tumor necrosis factor (TNF)-alpha, cytokine-induced neutrophil chemoattractant (CINC), expression of CD11b on the blood neutrophil cell surface, and neutrophil sequestration in multiple organs were examined 2 or 4 hr after the LPS challenge. Treatment of rats with HES (3.75 and 7.5 ml/kg) prevented LPS-induced NF-kappaB activation, and inhibited, in a dose-related manner, LPS-induced TNF-alpha and CINC expression. The 4 graded doses of HES decreased CD11b expression in a dose-dependent manner. HES significantly reduced neutrophil sequestration in lung, heart, and liver. These results suggest that HES has an anti-inflammatory effect in endotoxic rats. This effect is mediated by inhibition in the production pathways for inflammatory mediators, including NF-kappaB activation.

Animals↗

PubMed2002

Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157.

We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.

Amino Acid Sequence↗

PubMed2002

Glycosylation of beta(1)-adrenergic receptors regulates receptor surface expression and dimerization.

The beta(1)-adrenergic receptor (beta(1)AR) has one predicted site of N-linked glycosylation on its extracellular amino-terminus, but the glycosylation and potential functional importance of this site have not yet been examined. We show here that the beta(1)AR is glycosylated in various cell types and that mutation of the single predicted site of N-linked glycosylation (N15A) results in the formation of receptors that are not N-glycosylated. The beta(1)AR N15A mutant exhibited significantly decreased basal surface expression relative to the wild-type receptor but had no detectable deficits in ligand binding or agonist-promoted internalization. Co-immunoprecipitation experiments using Flag-tagged and HA-tagged receptors demonstrated that the beta(1)AR-N15A mutant receptor exhibits a markedly reduced capacity for dimerization relative to wild-type beta(1)AR. These data reveal that the beta(1)AR is glycosylated on Asn15 and that this glycosylation plays a role in regulating beta(1)AR surface expression and dimerization.

Amino Acid Substitution↗

PubMed2002

[Development of gene-viral vector and its anti-tumor effect, a primary study].

OBJECTIVE: To develop a new kind of vector system, named as gene-viral vector, which combines the advantages of the gene therapy and virus therapy. METHOD: An anti-tumor gene was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology. The killing effect, report gene expression of the green fluorescence protein, expression of the anti-tumor gene of mouse IL12, and the replication of the virus were observed respectively by cell pathology, fluorescence microscopy, ELISA and electron microscopy. RESULTS: A new kind of gene-viral vector system, in which the E1b-55 000 gene is deleted but the E1a gene of adenovirus is preserved, was constructed. The vector system possessed the same characteristics as the replicative virus ONYX-015, replication and proliferation in the tumor cells but not in the normal cells, thus specifically killing the tumor cells. Besides, it carried many kinds of anti-tumor genes. When carrying the report gene of the green fluorescence protein it made the expression of this gene in tumor cells far more effectively than the adenovirus vector employed in the traditional gene therapy did. However in the normal cells the expression of green fluorescence protein caused by this vector system was as little as or even less than that by the traditional adenovirus system. The similar result was also observed in the experiments of this vector system carrying the anti-tumor gene, gene of mouse IL12. The replication and proliferation of the virus carrying the gene of mouse IL12 in the tumor cells were confirmed by electron microscopy. CONCLUSION: Gene-viral vector is a new kind of vector in which the anti-tumor gene is inserted into the genome of the replicative virus specific for the tumor cells. It increases the expression of the anti-tumor gene by hundreds even tens of thousand times. It posseses all the advantages of gene therapy and virus therapy, thus further enhancing the curative effect and it overcomes such disadvantages as low transfer rate, low expression, lack of target tropism and low anti-tumor activity. It will become one of the most promising means in tumor treatment.

Adenoviridae↗

PubMed2002

Mechanisms of selective head cooling for resuscitating damaged neurons during post-ischemic reperfusion.

OBJECTIVE: To evaluate the efficacy and the mechanism of application of selective head cooling on neuronal morphological damage during postischemic reperfusion in a rabbit model. METHODS: 168 New Zealand rabbits were randomized into three groups. Group I [n = 24, (38 +/- 0.5) degrees C, non-ischemic control]; Group II [n = 72, (38 +/- 0.5) degrees C, normothermic reperfusion]; Group III [n = 72, (28 +/- 0.5) degrees C, selective head cooling, initiated at the beginning of reperfusion). Animals in three subgroups (n = 24, each) of Group II and Group III had reperfused lasting for 30, 180 and 360 min respectively. Using computerized image analysis technique on morphological changes of nucleus, the degree of neuronal damage in 12 regions were differentiated into type A (normal), type B (mild damaged), type C (severely damaged) and type D (necrotic). Fourteen biochemical parameters in brain tissues were measured. RESULTS: As compared with Group I, the counts of type A neuron decreased progressively, and those of type B, C and D increased significantly in Group II during reperfusion (P < 0.01). In Group II, vasoactive intestinal peptide, b-endorphine, prostacyclin, T3 and Na+, K(+)-ATPase were correlated with the changes of type A; b-endorphine and thromboxane with type B; glucose and vasopressin with type C; Na+, K(+)-ATPase, glutamic acid, T3 and vasoactive intestinal peptide with type D (P < 0.05). As compared with Group II, the counts of type A increased, and those of type C and D significantly decreased in Group III (P < 0.01). In Group III, Ca2+, Mg(2+)-ATPase were correlated with the changes of type A, C and D (P < 0.01). CONCLUSION: Selective head cooling for sex hours during postischemic reperfusion does improve neuronal morphological outcomes in terms of morphological changes.

Animals↗

PubMed2002

[Serological investigations on patients with hemolytic uremic syndromes due to enterohemorrhagic Escherichia coli O157:H7 infection].

OBJECTIVE: To investigate the etiological agent of patients with diarrhea followed by acute kidney failure symptoms in China, 1999. METHODS: Western blot was used to detect serum specific antibodies of patients against entero-haemorrhagic Escherichia coli hemolysin (EHEC-Hly) and lipo-polysaccharide of E. coli O157. RESULTS: Twenty-one and 16 of 42 patients showed positive reaction of specific IgG or IgM antibodies against EHEC-Hly respectively. Eleven of 42 serum samples were positive for having both IgG and IgM antibodies while 26 of 42 samples were positive for IgG or IgM. For E. coli O157 LPS test, 24 and 24 of 42 samples showed positive for IgG or IgM antibodies respectively. In 42 samples, 20 were positive for IgG and IgM while 29 were positive for IgG or IgM. CONCLUSIONS: Twenty-two of 42 samples were reacted with EHEC-Hly and E. coli O157 LPS, but 34 of 42 samples were positive for EHEC-Hly or E. coli O157. In combination of western blot results, bacterial isolation clinical symptoms and epidemiological investigation findings, it was reasonable to conclude that this cluster of patients with distinguish clinical symptoms was caused by E. coli O157:H7, which had never been reported in China. Hence serological methods with EHEC-Hly and E. coli O157 LPS are valuable for diagnosis of infections of E. coli O157:H7, when bacterial isolation is failed.

Adult↗

PubMed2002

[Study on diarrhea disease caused by enterohemorrhagic Escherichia coli O157:H7 in Xuzhou city, Jiangsu province in 2000].

OBJECTIVE: To investigate the proportion of hemorrhagic colitis (HC) caused by Escherichia coli O157:H7 in bacterial diarrhea in Xuzhou city, Jiangsu province. METHODS: All stool samples from patients with diarrhea were screened for O157 antigen, using Immuno-gold kits. Positive samples were cultured to detect the existence of pathogens. All of the HC patients confirmed by bacterial isolation and identification were investigated for clinical symptoms and laboratory tests. RESULTS: Of the diarrhea patients identified in Feng county in May, and in Tongshan county of Xuzhou city in June 2000, Jiangsu province 0.98% and 5.89% were caused by Escherichia coli O157:H7 respectively, confirmed by bacteriological isolation and identification of stool samples. At the early phase of hemorrhagic colitis, 18.5% patients had at least one abnormal clinical laboratory test results including protein in urea and increased BUN or creatinine that indicating the possibility of kidney damage. In 27 strains of E. coli O157:H7 isolated from those patients, 13 and 14 were identified as Shiga toxin producing and Shiga-toxin negative E. coli O157:H7 (Stx-positive or Stx-negative) respectively. By analysis of the two groups of patients divided by according to the nature of Shiga toxin, four of 13 patients of Stx-positive group showed positive urea protein. However only 1 of the 13 patients of Stx-negative group was urea protein positive. The decreased Platelets counts were observed in 6 of 13 patients with Stx-positive group, but only in 1 of 14 patients with stx-negative group. These differences were statistically significant. CONCLUSION: HC patients caused by E. coli O157:H7 were commonly seen (up to 5.89%) in Xuzhou city, Jiangsu province. Early laboratory tests should be conducted for HC patients as early as possible in order to find early indictor of kidney failure which was critical for prevention of hemolytic uremic syndrome.

Adolescent↗

PubMed2002

[Molecular typing of Shiga-toxin producing Escherichia coli O157:H7 isolated in China with pulsed field gel electrophresis].

OBJECTIVE: To type and group the Shiga-toxin producing Escherichia coli O157:H7 strains isolated recent years in China to understand the epidemiological features caused by the pathogen. METHODS: Pulsed field gel electrophoresis of large restriction fragments of bacterial chromosomal DNA was used. RESULTS: The 51 isolates of E. coli O157:H7 collected in recent years in China could be divided into 8 Pulsed Field Gel Electrophoresis (PFGE) types based on the size and number of restriction fragments and patterns, that were digested by XbaI. Strains isolated from Ningxia province showed only two types- PFGE1 and PFGE2. Strains isolated in Xuzhou of Jiangsu province had 6 PFGE types. Isolates identified between 1986 - 1988 belonged to PFGE7. Strains isolated from patients in 1999 - 2000 were PFGE5 and PFGE3. Strains isolated from stool samples of domestic animal, food and vegetable were PFGE3 - 6, of which the predominant type was PFGE5. All of the 5 strains isolated from patients with diarrhea and hemolytic uremic syndrome (HUS) belonged to PFGE type 5, which was the dominant type of the isolates from stool samples of domestic animal and samples of food and vegetable contaminated. CONCLUSION: Data suggested that the cluster patients with diarrhea and HUS might have been related to the pathogens from domestic animas and contaminated food or vegetables. The distribution of PFGE types also varied in different provinces of China.

Electrophoresis, Gel, Pulsed-Field↗

PubMed2002

Ketamine reduces NFkappaB activation and TNFalpha production in rat mononuclear cells induced by lipopolysaccharide in vitro.

Ketamine may be advantageous for anesthesia of patients with sepsis caused by gram-negative bacteria, because ketamine may suppress LPS-induced production of proinflammatory cytokines, such as TNFalpha and IL-6. NFkappaB is an important transcription factor that is involved in the post-transcriptional regulation of mRNA expression for several immunoinflammatory mediators in response to endotoxemia. This study examined the effect of ketamine on NFkappaB activation and TNFalpha production in rat peripheral blood mononuclear cells (PBMC). The PBMC were incubated in the presence or absence of LPS and with graded concentrations of ketamine. The culture supernatants and cells were collected for each group and duration of incubation. Activation of NFkappaB was determined by electrophoretic mobility shift assay (EMSA), and the expression of IkappaBalpha, its inhibitor, in PBMC was analysed by Western blotting. TNFalpha levels in the supernatants were measured using a specific enzyme-linked immunosorbent assay (ELISA). LPS stimulation of rat PBMC increased TNFalpha production and NFkappaB activation, with corresponding loss of IkappaBalpha. Ketamine significantly reduced the LPS-induced NFkappaB activation and inhibited TNFalpha production in a dose-dependent manner. These in vitro findings suggest that ketamine is a potent inhibitor of NFkappaB activation and cytokine production in rat PBMC.

Anesthetics, Dissociative↗

PubMed2002

Gene-viral vectors: a promising way to target tumor cells and express anticancer genes simultaneously.

OBJECTIVE: To develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies. METHODS: Using recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively. RESULTS: A new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy. CONCLUSIONS: Gene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

Adenoviridae↗

PubMedDate not supplied

Putative membrane assembly of EtpM-colicin V chimeras.

EtpM of the enterohemorrhagic E. coli O157:H7 is a bitopic membrane protein of the type II protein secretion apparatus. There is a twin-arginine (RR) motif in front of its signal anchor, suggesting a Tat-dependent membrane targeting of EtpM. By exploiting the periplasmic bactericidal activity of colicin V (ColV), we constructed EtpM-ColV fusions and studied the EtpM-mediated translocation of ColV. The wild type strain and the DeltatatC mutant were killed by the expressed fusions and were fully protected from the killing effect by the ColV-specific immunity protein. In contrast, cold-inactivation of YidC, which is generally required for integral membrane protein assembly, significantly attenuated the killing effect in the cold-sensitive yidC mutant. These results confirmed the predicted N(in)-C(out) EtpM topology, and suggests an EtpM-mediated, Tat-independent and YidC-dependent translocation of ColV.

Amino Acid Motifs↗