Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.     

Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products. With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. Copyright 1998 John Wiley & Sons, Inc.     

Identification of a negative regulator for the pathogenicity island of enterohemorrhagic<Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

EnterohemorrhagicEscherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue. The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island. The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons,LEE1,LEE2,LEE3,LEE4, andtir (LEE5). The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing. Current knowledge is that regulation is hierarchical: a pivotal positive regulator,ler, is first stimulated, which in turn activates the expression of other operons. Herein, we report on the presence of a negative regulation protein located within the LEE.L0044 is 372 bp in length and is located outside of the 5 major operons. An isogenicL0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins. Reciprocally,trans expression ofL0044 suppressed the expression of the LEE. Furthermore, mRNA ofler increased as a result of deletingL0044, and disruptingler in aL0044-deleted background reversed the loss of the repression phenotype. Thus,L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation ofler.     

Differential distribution of proteins expressed in companion cells in the sieve element-companion cell complex of rice plants

Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.     

Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis

Antibodies to citrullinated proteins (anti-cyclic-citrullinated peptide [anti-CCP] antibodies) are highly specific for rheumatoid arthritis (RA) and precede the onset of disease symptoms, indicating a pathogenetic role for these antibodies in RA. We recently showed that distinct genetic risk factors are associated with either anti-CCP-positive disease or anti-CCP-negative disease. These data are important as they indicate that distinct pathogenic mechanisms are underlying anti-CCP-positive disease or anti-CCP-negative disease. Likewise, these observations raise the question of whether anti-CCP-positive RA and anti-CCP-negative RA are clinically different disease entities. We therefore investigated whether RA patients with anti-CCP antibodies have a different clinical presentation and disease course compared with patients without these autoantibodies. In a cohort of 454 incident patients with RA, 228 patients were anti-CCP-positive and 226 patients were anti-CCP-negative. The early symptoms, tender and swollen joint count, and C-reactive protein level at inclusion, as well as the swollen joint count and radiological destruction during 4 years of follow-up, were compared for the two groups. There were no differences in morning stiffness, type, location and distribution of early symptoms, patients' rated disease activity and C-reactive protein at inclusion between RA patients with and without anti-CCP antibodies. The mean tender and swollen joint count for the different joints at inclusion was similar. At follow-up, patients with anti-CCP antibodies had more swollen joints and more severe radiological destruction. Nevertheless, the distribution of affected joints, for swelling, bone erosions and joint space narrowing, was similar. In conclusion, the phenotype of RA patients with or without anti-CCP antibodies is similar with respect to clinical presentation but differs with respect to disease course.     

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.     

Visualization of the structures of the hepatitis C virus replication complex

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.     

A portable potentiostat for the bilirubin-specific sensor prepared from molecular imprinting

A portable amperometric potentiostat was designed and implemented in this work. It was developed to acquisit the current signals produced from bilirubin by an electrochemical sensor. Based on an SOC-based chip, this potentiostat has the merits of moderate accuracy, small size, low cost, and high portability. The bilirubin electrode was prepared by synthesizing a thin layer of bilirubin imprinted poly(methacrylic acid-co-ethylene glycol dimethacrylate) onto the Au layer. With the molecularly imprinted polymer (MIP) film, specific detection of bilirubin was successfully achieved. The cyclic voltammogram of the electrode was measured from this assembled potentiostat. The performance from a commercial potentiostat was considered rather stable and was used as a reference to examine and evaluate the performance of the assembled potentiostat. The detected current signals by the bilirubin sensing were obtained. Linear calibration with a sensitivity of 1.344+/-0.38 microA/mg dl was achieved. Our experimental results showed that the proposed potentiostat's performance could achieve sufficient performance. The evaluation was also made from the aspects such as reset time and steady-response time. The self-assembled potentiostat thus demonstrated its ability in precise detection of bilirubin from an electrode layered with the imprinted polymer film.