Phase 1 trial of intraperitoneal paclitaxel in combination with intravenous cisplatin and oral capecitabine in patients with advanced gastric cancer and peritoneal metastases (IPGP study).

AIMS: Gastric cancer with peritoneal involvement has a poor prognosis. Intraperitoneal (IP) paclitaxel has shown promising results in these patients. However, this approach has only been studied in the Asian population, and in combination with S-1. We investigated the maximum tolerated dose of IP paclitaxel, with a standard chemotherapy combination, in the Australian population. METHODS: The study of the population included metastatic human epidermal growth factor receptor 2 (HER2) negative gastric adenocarcinoma with peritoneal involvement. Treatment included six 21-day cycles of cisplatin (80 mg/m2 IV, day 1) plus capecitabine (1000 mg/m2 PO BD, days 1-14) plus IP paclitaxel (days 1 and 8). IP paclitaxel doses for cohort 1-3 were 10, 20, and 30 mg/m2 , respectively, in a 3 + 3 standard dose-escalation design. RESULTS: Fifteen patients were enrolled of which 6 were female and the median age was 63. Two patients developed dose-limiting toxicities. No grade 4/5 toxicities were recorded. The maximum tolerated dose was not reached. Therefore, as defined by the study protocol, the recommended phase-2 dose for IP paclitaxel was determined to be 30 mg/m2 . The 12-month survival rate was 46.7%, and the median survival was 11.5 months (interquartile range [IQR]: 15.3-6.9). CONCLUSIONS: IP paclitaxel is safe in combination with cisplatin and capecitabine and the recommended phase-2 dose is 30 mg/m2 .

Hypoxia-Activated, Small-Molecule-Induced Gene Expression.

Hypoxia, a condition of reduced oxygen, occurs in a wide variety of biological contexts, including solid tumors and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated, small-molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-ß-d-galactopyranoside. As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production of any given protein of choice.

[Auto-inducible expression system based on the SigB-dependent ohrB promoter in Bacillus subtilis].

The reliable production of heterologous proteins is important in the field of industrial biotechnology. This can be achieved by applying auto-inducible gene expression systems. The development of a Bacillus subtilis expression plasmid harboring SigB-dependent ohrB promoter was reported. The expression system was subjected to high cell density cultivation to produce xylanase as a stable model protein. The recombinant strain was cultured in a synthetic medium containing glucose as the carbon source. The exponential fed-batch feeding strategy was applied to prevent substrate inhibition. A sharp increase of xylanase activity (about 6-fold) at the end of the fermentation was observed as a result of sigma factor B (SigB) protein activation, supporting auto-inducibility of the expression system. For the control strain a specific induction of the xylanase activity was not observed. The recombinant strain was capable to offer a 5-fold increase in xylanase activity in comparison with the control strain. In addition, the constructed system displayed catabolite repression resistance ability. This SigB-dependent expression system can be considered as a biotechnology tool and an alternative to eliminate the cost of conventional inducers, e.g. isopropyl-ß-galactopyranoside.

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

UNLABELLED: Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-ß-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. SIGNIFICANCE AND IMPACT OF THE STUDY: A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.

E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification.

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, activation-induced deaminase. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged Ig lambda(R) gene in the chicken DT40 B cell line, which performs constitutive Ig gene diversification. By analysis of a DT40 derivative in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that E2A must function in cis to promote diversification and that stimulation of diversification in cis depends on the E2A activation domains. By direct imaging, we show that lambda(R)/E2A colocalizations are most prominent in G(1). We further show that expression of the E2A antagonist Id1 prevents lambda(R)/E2A colocalizations in G(1) and impairs diversification but not transcription of lambda(R). Thus, E2A acts in cis to promote Ig gene diversification, and G(1) phase is the critical window for E2A action.

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered Escherichia coli and Saccharomyces cerevisiae cells.

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as "enzyme bags" and incubated at 30 degrees C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 microg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26 degrees C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.

Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli.

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor.

Various host-vector combinations were tested to maximize the extracellular production of recombinant asparaginase in Escherichia coli. Expression of recombinant asparaginase fused to pelB leader sequence under the inducible T7lac promoter in BLR (DE3) host cells resulted in optimum extracellular production in shake-flasks. Fed-batch studies were carried out using this recombinant strain and an exponential feeding strategy was used to maintain a specific growth rate of 0.3 h(-1). To check the effect of the time of induction on expression, cultures were induced with 1 mM isopropyl-beta-D-thiogalactopyranoside at varying cell optical densities (OD(600): 33, 60, 90, 135). Although the specific product formation rates declined with increasing OD of induction, a maximum volumetric activity of 8.7 x 10(5) units l(-1), corresponding to approximately 5.24 g l(-1) of recombinant asparaginase, was obtained when induction was done at an OD(600) of 90. The recombinant protein was purified directly from the culture medium, using a rapid two-step purification strategy, which resulted in a recovery of approximately 70% and a specific activity of approximately 80% of that of the native enzyme.

Visualization of the dynamics of gene expression in the living mouse.

Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals. We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse. Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus. The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal.

Bacterial expression, purification, and characterization of rat kidney-type mitochondrial glutaminase.

The human gene that encodes the kidney-type glutaminase (KGA) spans 84-kb, contains 19 exons, and encodes two alternatively spliced mRNAs. Various segments of the rat KGA cDNA were PCR amplified and cloned into a bacterial expression vector to determine whether the N- and C- terminal ends of the glutaminase protein were essential for activity. A recombinant glutaminase, lacking the coding sequence contained in exon 1, was found to be fully active. In contrast, proteins that lacked sequences from exons 1 and 2 and exons 1-3 were inactive. An additional construct that corresponded to the sequence encoded by exons 2-14 also retained full activity. Both of the fully active, truncated proteins were purified to apparent homogeneity using an incorporated N-terminal His(6)-tag and Ni(2+)-affinity chromatography. The K(M) values for glutamine of the native and recombinant forms of glutaminase were nearly identical. However, the two truncated forms of the glutaminase exhibit the characteristic phosphate activation profile only when dialyzed into a buffer lacking phosphate. Dialysis versus 10mM Tris-phosphate was sufficient to form an active tetramer. Thus, the deleted N-terminal sequence may contribute to the phosphate-dependent oligomerization and activation of the native glutaminase.

Mapping stress-induced changes in autoinducer AI-2 production in chemostat-cultivated Escherichia coli K-12.

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-beta-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.

Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.

Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flärdh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

An in vivo study of novel bioactive peptides that inhibit the growth of Escherichia coli.

We have created a system in which synthetically produced novel bioactive peptides can be expressed in vivo in Escherichia coli. Twenty thousand of these peptides were screened and 21 inhibitors were found that could inhibit the growth of E. coli on minimal media. The inhibitors could be placed into one of two groups, 1-day inhibitors, which were partially inhibitory, and 2-day inhibitors, which were completely inhibitory. Sequence analysis showed that two of the most potent inhibitors were actually peptide-protein chimeras in which the peptides had become fused to the 63 amino acid Rop protein which was also contained in the expression vector used in this study. Given that Rop is known to form an incredibly stable structure, it could be serving as a stabilizing motif for these peptides. Sequence analysis of the predicted coding regions from the next 10 most inhibitory peptides showed that four of the 10 peptides contained one or more proline residues either at or very near the C-terminal end of the peptide which could act to prevent degradation by peptidases. Collectively, based on what we observed in our screen of synthetic bioactive peptides that could prevent the growth of E. coli and what has been learned from structural studies of naturally occurring bioactive peptides, the presence of a stabilizing motif seems to be important for small peptides, if they are to be biologically active.

[Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]. / Reguliatsiia sinteza beta-galaktozidazy Escherichia coli ékzogennym tsiklicheskim 3',5'-adenozinmonofosfatom.

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.