Persistence of foetal anastomoses between carotid and vertebrobasilar arteries is well recognised, but their bilateral presence is rare. We report a case of bilateral persistent hypoglossal arteries. Absence of bilateral vertebral arteries was incidentally noted on neck ultrasound examination. Subsequent MRI confirmed this and revealed persistent hypoglossal arteries that arose bilaterally from the cervical internal carotid artery, entered the cranium through the hypoglossal canal and joined the lower portion of the basilar artery.
Although the oxidative stress frequently occurs in patients with chronic hepatitis C, its role in future hepatocellular carcinoma (HCC) development is unknown. Hepatic 8-hydroxydeoxyguanosine (8-OHdG) was quantified using liver biopsy samples from 118 naïve patients who underwent liver biopsy from 1995 to 2001. The predictability of 8-OHdG for future HCC development and its relations to epidemiologic, biochemical and histological baseline characteristics were evaluated. During the follow-up period (mean was 6.7±3.3 years), HCC was identified in 36 patients (30.5%). Univariate analysis revealed that 16 variables, including 8-OHdG counts (65.2±20.2 vs 40.0±23.5 cells per 105 μm2, P<0.0001), were significantly different between patients with and without HCC. Cox proportional hazard analysis showed that the hepatic 8-OHdG (P=0.0058) and fibrosis (P=0.0181) were independent predicting factors of HCC. Remarkably, 8-OHdG levels were positively correlated with body and hepatic iron storage markers (vs ferritin, P<0.0001 vs hepatic iron score, P<0.0001). This study showed that oxidative DNA damage is associated with increased risk for HCC and hepatic 8-OHdG levels are useful as markers to identify the extreme high-risk subgroup. The strong correlation between hepatic DNA damage and iron overload suggests that the iron content may be a strong mediator of oxidative stress and iron reduction may reduce HCC incidence in patients with chronic hepatitis C.
oxidative stress; free radicals; 8-hydroxydeoxyguanosine; iron; hepatitis C virus; immunohistochemistry
In a previous study, we showed that levels of cell-free DNA (cfDNA) were significantly higher in sera of patients with hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV) than in sera of non-HCC patients with HCV. To confirm this finding, we analysed serum cfDNA levels in a cohort of 96 patients with HCV-related HCC and in 100 HCV carriers without known HCC. Again we found that serum cfDNA levels were significantly higher in HCC patients than in HCV carriers (115.9±98.3 vs 34.4±40.4 ng ml−1 (mean±s.d.), P<0.0001). Of 87 eligible patients who underwent curative hepatectomy, those with a high cfDNA level had a significantly shorter overall survival (OS) time than those in whom the cfDNA level was not high. Cox proportional hazards model showed the cfDNA level to be an independent prognostic factor for OS and cancer recurrence in distant organs. Our results suggest that the serum cfDNA level reflects the metastatic potential of HCV-related HCC and that it can be a useful predictive biomarker for distant metastasis after curative surgery.
HCV; HCC; blood testing; cell-free DNA; real-time PCR; GSTP1
Rett syndrome is a neurodevelopmental disorder with severe mental retardation caused by mutations in the MECP2 gene. Mutations in the MECP2 gene are also associated with other genetic disorders, including X linked mental retardation in males. Missense mutations identified so far are present primarily in the methyl CpG binding domain (MBD) of MECP2. Here, the functional significance of 28 MBD missense mutations identified in patients were analysed by transient expression of the mutant proteins in cultured cells. The effects of mutations were evaluated by analysis of the affinity of MeCP2 to pericentromeric heterochromatin in mouse L929 cells and on transcriptional repressive activity of MeCP2 in Drosophila SL2 cells. These analyses showed that approximately one-third (9/28) of MBD missense mutations showed strong impairment of MeCP2 function. The mutation of the R111 residue, which directly interacts with the methyl group of methyl cytosine, completely abolished MeCP2 function and mutations affecting ß-sheets and a hairpin loop have substantial functional consequences. In contrast, mutations that showed marginal or mild impairment of the function fell in unstructured regions with no DNA interaction. Since each of these mutations is known to be pathogenic, the mutations may indicate residues that are important for specific functions of MeCP2 in neurones.
We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction. The protein was identified as one of the two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning. These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively. The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases. The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II. They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins. The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined. The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein.
To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the alpha-subunit of RNA polymerase in TyrR-mediated activation of the mtr promoter, we have carried out in vitro transcription experiments using a set of mutant RNA polymerases with a supercoiled mtr template. Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K297A suggested the presence of a possible UP element associated with the mtr promoter. Mutational studies have revealed that an AT-rich sequence centered at -41 of the mtr promoter (SeqA) functions like an UP element. In vivo and in vitro analyses using a mutant mtr promoter carrying a disrupted putative UP element showed that this AT-rich sequence is responsible for interactions with the alpha-subunit which influence transcription in the absence of TyrR protein. However, the putative UP element is not needed for activator-dependent activation of the mtr promoter by TyrR and phenylalanine. The results from in vitro studies indicated that the alpha-subunit residues leucine-262, arginine-265, and lysine-297 are critical for interaction with the putative UP element of the mtr promoter and play major roles in TyrR-dependent transcription activation. The residues at positions 258, 260, 261, 268, and 270 also play important roles in TyrR-dependent activation. Other residues, at positions 259, 263, 264, 266, 269, 271, 273, 275, and 298, appear to play less significant roles or no role in activation of mtr transcription.
Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflammation, has been reported to induce expression of the gene for type II inducible nitric oxide synthase (iNOS) in peripheral organs. This study was carried out to examine whether intraperitoneally injected LPS elicits the expression of iNOS messenger ribonucleic acid (mRNA) in the rat brain. We also investigated whether intraperitoneal treatment with dexamethasone (DEX) prevents this induction. To determine levels of iNOS mRNA, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was employed. Treatment with LPS induced the expression of iNOS mRNA in various brain regions, accounting for approximately 1 x 10(5) to 4 x 10(5) molecules per micrograms of poly A+ RNA, and these inductions were markedly suppressed by DEX. The results suggest that, during systemic inflammation, iNOS mRNA induction occurs in brain through a DEX-sensitive mechanism.
Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1 and P2. Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2 were studied with a P1-mutant. We have identified the precise location of alpha binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase alpha subunit: alpha binds to different sites at the upstream end of each complex. Transcription initiation at galP1 can be activated by the cyclic AMP receptor protein (CRP). Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of alpha, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1 complex. Thus, the C-terminal domain of alpha can occupy three different sites at the gal operon regulatory region. We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of alpha in ternary CRP-RNA polymerase-galP1 complexes. Footprinting experiments show that these substitutions interfere with the contact between CRP and alpha but do not affect the position of alpha binding to its site upstream of bound CRP.
The Rob protein, isolated on the basis of its ability to bind to the right arm of the Escherichia coli origin of chromosomal replication, is about 50% identical in amino acid sequence to SoxS and MarA, the direct regulators of the superoxide (soxRS) and multiple antibiotic resistance (mar) regulons, respectively. Having previously demonstrated that SoxS (as a MalE-SoxS fusion protein) and MarA are essentially identical in their abilities to activate in vitro transcription of genes of the sox-mar regulons, we investigated the properties of Rob as a transcriptional activator. We found that Rob (i) activates the transcription of zwf,fpr,fumC, micF, nfo, and sodA, (ii) requires a 21-bp soxbox-marbox-robbox sequence to activate zwf transcription, (iii) protects the soxbox/marbox/robbox from attack by DNase 1, (iv) is ambidextrous, i.e., requires the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF, (v) bends zwf and fumC DNA, and (vi) binds zwf and fumC DNA as a monomer. Since these transcription activation properties of Rob are virtually identical to those of MalE-SoxS and MarA, it appears as if the E. coli genome encodes three genes with the same functional capacity. However, in contrast to SoxS and MarA, whose syntheses are induced by specific environmental stimuli and elicit a clear defense response, Rob is expressed constitutively and its normal function is unknown.
The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase alpha subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the alpha subunit. To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3' half of E. coli rpoA, the gene encoding the alpha subunit of RNA polymerase. We isolated a number of rpoA mutants defective in oxyR-dependent transcription activation at the E. coli katG promoter. Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the alpha subunit. Reconstituted RNA polymerases containing the mutant alpha subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promoters by OxyR. These results suggest that these two regions comprise the contact surfaces on the alpha subunit for OxyR.
Expression of the marA or soxS genes is induced by exposure of Escherichia coli to salicylate or superoxides, respectively. This, in turn, enhances the expression of a common set of promoters (the mar/soxRS regulons), resulting in both multiple antibiotic and superoxide resistance. Since MarA protein is highly homologous to SoxS, and since a MalE-SoxS fusion protein has recently been shown to activate soxRS regulon transcription, the ability of MarA to activate transcription of these genes was tested. MarA was overexpressed as a histidine-tagged fusion protein, purified, cleaved with thrombin (leaving one N-terminal histidine residue), and renatured. Like MalE-SoxS, MarA (i) activated the transcription of zwf, fpr, fumC, micF, nfo, and sodA; (ii) required a 21-bp "soxbox" sequence to activate zwf transcription; and (iii) was "ambidextrous," i.e., required the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF. Thus, the mar and soxRS systems use activators with very similar specificities and mechanisms of action to respond to different environmental signals.
Two hundred forty-five consecutive amino acids of the sigma 70 subunit of Escherichia coli RNA polymerase are not conserved in the homologous protein of Bacillus subtilis. We show that their deletion from a sigma 70-32 hybrid protein caused no severe loss of function in vivo, while sigma 70 itself retained considerable function in vitro.
A number of transcription activators have been found to activate transcription via protein-protein contact between RNA polymerase alpha subunits and transcription factors; they are classified as class I factors. In this report, we demonstrate that the FlhD/FlhC complex, a transcription activator of the Escherichia coli flagellar regulon, requires the C-terminal domain of the RNA polymerase alpha subunit for transcription activation. We conclude that FlhD/FlhC is a class I transcription factor.
The purified TyrR protein and phenylalanine were sufficient to activate in vitro transcription from the tyrP promoter by wild-type RNA polymerase. Such TyrR-mediated activation did not occur when the mutant alpha 235 RNA polymerase was used, indicating that TyrR is a class I transcription activator.
A girl with intractable epilepsy who received high dose valproic acid showed bone marrow suppression, which improved after the end of valproic acid treatment and the administration of supportive treatments. Valproic acid markedly suppressed the in vitro colony growth in this patient. It is believed that high dose valproic acid treatment directly suppressed the growth of haematopoietic progenitors, resulting in bone marrow failure.
The chromosomal DNA fragments of Escherichia coli K-12 were cloned into a mini-F cosmid, pRE435, after partial digestion with restriction endonuclease Sau3AI. The clones were first screened for PyrC+ and then for other genes, including rpmF encoding ribosomal protein L32 that had been mapped near pyrC (I. Janda, M. Kitakawa, and K. Isono, Mol. Gen. Genet. 201:443-436, 1985). Thus, we obtained a total of five rpmF-containing clones. The rpmF gene was located on the chromosomal segment in one of the clones (pAY2-5) by insertional mutagenesis with transposon gamma delta, followed by analysis of the gene products by the maxicell method. Hybridization analysis of clone pAY2-5 with the ordered clone bank (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) indicates that a gap at the 1,510-kilobase coordinates in the physical map of E. coli can be bridged by this clone. The nucleotide sequence of the region containing rpmF was accordingly established. In addition, the RNA transcripts from the chromosomal region containing rpmF were analyzed, and the transcriptional initiation sites were determined. The results suggest that rpmF forms an operon with the gene termed g30k which codes for a 30-kilodalton protein of unknown function. At least four transcripts were found to code for ribosomal protein L32.
Our recent clinical experience suggested that bacteremia may interfere with the detection of concomitant fungemia when standard blood culture methods are used. To determine the extent to which bacteria may interfere with fungal isolation from blood cultures, an in vitro model simulating blood cultures taken during concomitant fungemia and bacteremia was created. Each of six bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) was combined with each of three pathogenic yeasts (Candida albicans, Candida tropicalis, and Torulopsis glabrata) in vented blood culture bottles containing enriched brain heart infusion broth and fresh normal human blood. Blood culture bottles were analyzed at 1, 2, and 7 days of incubation. Gram strains and subcultures onto chocolate and MacConkey agars failed to detect fungi in 37.0, 66.7, and 100% of samples, respectively. However, subcultures onto Sabouraud dextrose agar failed in only 13% of the samples (occurring only with P. aeruginosa). In a rabbit model of concomitant fungemia with C. albicans and bacteremia with P. aeruginosa, no yeasts were recovered from blood cultures despite 100% detection of P. aeruginosa. Therefore, the usual microbiological techniques may be inadequate to detect fungemia when concomitant bacteremia is present.
The combination of amphotericin B and rifampin was synergistic in vitro in both inhibiting and killing seven strains of Cryptococcus neoformans by the checkerboard microtitration technique.
In order to find the promoter region of phosphoenolpyruvate carboxylase [EC 126.96.36.199] gene (ppc), in vitro transcription was performed using truncated DNA fragments as templates. Transcription mapping showed three promoters as candidates, but only one of them could be assigned to the promoter of ppc gene, considering the nucleotide sequence of its coding region (Fujita, N., Miwa, T., Ishijima, S., Izui, K. and Katsuki, H. (1984) J. Biochem. 95, 909-916). Nuclease S1 mapping showed that the in vivo and in vitro transcription initiation sites are identical and that the site lies 91 or 92 nucleotides upstream the translation initiation site. No alteration of the transcription initiation site was observed whether the cells were starved for an amino acid or grown on various carbon sources. The sequences of the -10 and -35 regions were fairly in accordance with the consensus sequences hitherto reported. Some features of the sequence around the promoter region were discussed.
Sequence determinants responsible for promoter recognition by RNA polymerase holoenzyme containing sigma 38, the rpoS gene product, were analyzed. In a previous study [Tanaka et al. (1993) Proc. Natl. Acad. Sci. USA, 90, 3511-3515], Escherichia coli promoters were classified into three groups: promoters recognized only by RNA polymerase holoenzyme containing sigma 70 (E sigma 70); promoters recognized preferentially by that containing sigma 38 (E sigma 38); promoters recognized by both E sigma 70 and E sigma 38. As representatives of each group of promoter, we chose the alaS, fic and lacUV5 promoters. Making use of a restriction enzyme site inserted between the -10 and -35 hexamer sequences, promoters were divided into the upstream (UE) and downstream (DE) elements. These UEs and DEs were combined in all possible combinations and used for in vitro transcription reactions. Promoters containing DE from the fic or lacUV5 promoter were found to be recognized by E sigma 38, while those containing DE from the alaS promoter were not. Moreover, fic DE alone functioned as an efficient promoter for E sigma 38. Thus we conclude that the discrimination signal resides within the DE sequence. To test the activator response of E sigma 38, in vitro transcription reactions were also performed with the gal and lac promoters. For both CRP-responsive P1 promoters, E sigma 38 was found to be activated by the CRP-cAMP complex.
An in vitro mixed transcription system was employed to examine the possible alteration of the promoter selectivity of Escherichia coli RNA polymerase by specific tRNAs. Transcription in vitro was inhibited by most of the tRNAs examined, although the extent of the inhibition differed with the tRNA species. The inhibition by tRNAs was due to competition with DNA for binding RNA polymerase. This inhibitory effect remained after charging of the tRNAs with amino acids. The charging of tRNAfMet with fMet, but not with Met, abolished its inhibitory effect, and instead gave a stimulatory effect on the transcription from some promoters. These observations suggest that fMet-tRNAfMet plays a specific regulatory role in the coupling of transcription to translation.