Efficacy and safety of percutaneous transhepatic portal embolization before right liver resection using an ethibloc/lipiodol mixture: a single-center experience.

AIM: The purpose of this study was to evaluate the safety and efficacy of percutaneous transhepatic portal vein embolization of the right portal vein with an Ethibloc/Lipiodol mixture to induce hypertrophy of the left liver lobe in patients with primarily unresectable liver tumor. METHODS: 15 patients (8 primary liver tumors, 7 liver metastases) underwent portal vein embolization. Liver volumetry, duration of hospitalization, complication rates, relevant laboratory values were documented. RESULTS: In 13/15 patients (84.6%) embolization could be performed with a median of 8.8 ml (range 1.5-28 ml) Ethibloc/Lipiodol. One minor procedure-related complication (subcapsular hematoma) occurred, which did not affect the two-step liver resection. No patient developed acute liver failure after embolization or liver resection. The volume of the left liver lobe increased significantly (p = 0.0015) by 25% from a median of 750 ml (587-1,114 ml) to 967 ml (597-1,249 ml). 11/13 (81.8%) of the embolized patients underwent liver resection at a median of 49 days after embolization. Median hospitalization time was 4 days after embolization and 7 days after liver resection. Median overall survival of the 11 operated patients was 376 days. CONCLUSION: Percutaneous transhepatic portal vein embolization using an Ethibloc/Lipiodol mixture is a safe, feasible, and efficient interventional procedure.

Transcriptional control of genes encoding CS1 pili: negative regulation by a silencer and positive regulation by Rns.

The adherence of enterotoxigenic Escherichia coli (ETEC) to the human small intestine is an important early event in infection. Attachment is thought to be mediated by proteinaceous structures called pili. We have investigated the regulation of expression of the genes encoding CS1 pili found on human ETEC strains and find that there are at least three promoters, P1 and P2, upstream of the coo genes, and P3, downstream of the start of cooB translation. We identified a silencer of transcription which extends over several hundred bases overlapping the cooB open reading frame. This silencer is dependent on the promoter and/or upstream region for its negative effect. The DNA binding protein H-NS is a repressor of coo transcription that acts in the same region as the silencer, so it is possible that H-NS is involved in this silencing. Rns, a member of the AraC family, positively regulates transcription of the coo operon and relieves the silencing of CS1 expression.

Genes for CS2 pili of enterotoxigenic Escherichia coli and their interchangeability with those for CS1 pili.

We have cloned and sequenced the DNA needed for production of CS2 pili in Escherichia coli K-12. The four open reading frames, cotB, cotA, cotC, and cotD, show homology with the genes needed for production of CS1 and CFA/I pili, which are also found on enterotoxigenic E. coli associated with human diarrheal disease. We also report that CotA plus CotB interact with the CS1 gene products CooC and CooD to form pili that can be visualized by electron microscopy and, conversely, that the CS1 gene products CooA and CooB interact with CotC and CotD to form pili.

Regulation of rns, a positive regulatory factor for pili of enterotoxigenic Escherichia coli.

Attachment of enterotoxigenic Escherichia coli to the human gut is considered an important early step in infection that leads to diarrhea. This attachment is mediated by pili, which belong to a limited number of serologically distinguishable types. Many of these pili require the product of rns, or a closely related gene, for their expression. We have located the major promoter for rns and found that although its sequence diverges significantly from a sigma-70 promoter consensus sequence, it is very strong. Transcription of rns is negatively regulated both at a region upstream of this promoter and at a region internal to the rns open reading frame. In addition, rns positively regulates its own transcription, probably by counteracting these two negative effects.

CooC and CooD are required for assembly of CS1 pili.

Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple identical protein subunits encoded by cooA. We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA. Following cooD is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E. coli K-12 to synthesize CS1 pili. Complementation analysis of mutants cloned in E. coli K-12 and constructed in an ETEC-derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.

CooB is required for assembly but not transport of CS1 pilin.

CS1 pili are filamentous proteinaceous appendages found on many enterotoxigenic Escherichia coli (ETEC) strains isolated from human diarrhoeal disease. They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells. We have identified a gene, cooB, which lies directly upstream of cooA, the gene that encodes the major structural CS1 protein. When translated in vitro, the protein product of cooB migrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence. We constructed a mutant allele (cooB-1) by insertion of the omega fragment, which inhibits transcription and translation, into the cooB gene in vitro. In a derivative of an ETEC strain with the cooB-1 mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, no cooB and a greatly reduced level of cooA product was detectable in total cell extracts. The reduction of cooA in this strain appears to result from polarity of the cooB mutation because introduction of the wild-type cooA gene in trans causes production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant. Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes. However, the cooB-1 strain with cooA in trans does not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili).(ABSTRACT TRUNCATED AT 250 WORDS)

A single-copy promoter-cloning vector for use in Escherichia coli.

We have constructed and tested a single-copy-plasmid vector (pEU720) based on the IncFII-group plasmid, R100, that is useful for cloning promoters in front of lacZ. The vector is 15 kb long and contains a unique XhoI site in front of lacZ.

Second-site revertants of the P1 copN22 copy mutant.

Miniplasmids with the P1 copN22 mutation have a copy number about seven times that of the wild type. Selection for reduced copy number from this plasmid led to the isolation of second-site pseudorevertants, called poc mutants. DNA sequence analysis showed that all six independent poc mutants have a single base change in the same codon of the repA gene. This implicates the amino acid at this location, either directly or indirectly, in interactions important for copy number control.

A single amino acid difference between Rep proteins of P1 and P7 affects plasmid copy number.

P1 and P7 are closely related plasmid prophages which are members of the same incompatibility group. We report the complete DNA sequence of the replication region of P7 and compare it to that of P1. The sequence predicts a single amino acid difference between the RepA proteins of these two plasmids, no differences in methylation sites or regions where dnaA protein is expected to bind, and no difference in the spacing of the major features of the two replicons. A P1 replicon with a mutation in repA, the gene that encodes an essential replication protein, is complemented for replication by providing either the P1 RepA protein (RepA1) or the P7 RepA protein (RepA7) in trans. Furthermore, when either of these proteins is supplied in trans, the plasmid copy number of P1 cop mutants drops to that of P1 cop+. However, when RepA7 is supplied, the copy number of P1 cop and P1 cop+ is higher than that when RepA1 is supplied. This indicates that the single amino acid difference between the two versions of the RepA protein plays an important role in determining the plasmid copy number.

IS1-dependent generation of high-copy-number replicons from bacteriophage P1 Ap Cm as a mechanism of gene amplification.

Mutant P1 Ap Cm lysogens were isolated in which the drug resistance genes resident on the plasmid prophage P1 Ap Cm are amplified by a novel mechanism. The first step required for amplification is IS1-mediated rearrangement of the P1 Ap Cm prophage. The drug resistance genes are amplified from the rearranged P1 Ap Cm prophage by the formation of a plasmid (P1dR) which contains the two resistance genes. The P1dR plasmid is an independent replicon about one-half the size of P1 Ap Cm that can be maintained at a copy number eightfold higher than that at which P1 Ap Cm can be maintained. It contains no previously identified replication origin and is dependent on the Rec+ function of the host.

Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants.

Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.

Escherichia coli mutants in which transcription is dependent on recA function.

A gene essential for viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdg for recA-dependent growth, is near 16 min on the E. coli chromosome between phr and gltA and is 90% cotransduced with gltA. In a strain with an rdg deletion and the temperature-sensitive recA allele, recA200, growth stopped within 7 min after cells were shifted to the nonpermissive temperature (42 degrees C). The cells remained viable for many hours at 42 degrees C. The defect at the nonpermissive temperature is in ribonucleic acid synthesis, which was completely shut off within 20 min after the temperature shift. Protein synthesis was also shut off, but deoxyribonucleic acid synthesis continued for at least 2 h after the shift. The rdg mutation alone had no apparent effect on growth, deoxyribonucleic acid repair, or recombination.

Weigle reactivation of the single-stranded DNA phage f1.

The survival of ultraviolet light (UV) damaged single-stranded DNA bacteriophage f1 is increased when the Escherichia coli host is irradiated with UV prior to infection. This repair, called Weigle reactivation, is multiplicity independent and is absent in recA and in lexA mutants. The function of the recA-lexA repair system needed is repair and not recombination, as demonstrated by the absence of Weigle reactivation in mutants that are recombination proficient but defective in repair of double-stranded DNA. Weigle reactivation of f1 requires high levels of the recA protein, and in addition activation of recA or another protein. This activation can be produced by UV irradiation, or by the tif-1 allele of recA together with the spr allele of lexA. Mutagenesis of f1 has the same requirements as W-reactivation, and in addition requires UV irradiation of the phage.