Simultaneous biodegradation of three mononitrophenol isomers by a tailor-made microbial consortium immobilized in sequential batch reactors.

The ortho-nitrophenol (ONP)-utilizing Alcaligenes sp. strain NyZ215, meta-nitrophenol (MNP)-utilizing Cupriavidus necator JMP134 and para-nitrophenol (PNP)-utilizing Pseudomonas sp. strain WBC-3 were assembled as a consortium to degrade three nitrophenol isomers in sequential batch reactors. Pilot test was conducted in flasks to demonstrate that a mixture of three mononitrophenols at 0·5 mol l-1 each could be mineralized by this microbial consortium within 84 h. Interestingly, neither ONP nor MNP was degraded until PNP was almost consumed by strain WBC-3. By immobilizing this consortium into polyurethane cubes, all three mononitrophenols were continuously degraded in lab-scale sequential reactors for six batch cycles over 18 days. Total concentrations of ONP, MMP and PNP that were degraded were 2·8, 1·5 and 2·3 mol l-1 during this time course respectively. Quantitative real-time PCR analysis showed that each member in the microbial consortium was relatively stable during the entire degradation process. This study provides a novel approach to treat polluted water, particularly with a mixture of co-existing isomers. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitroaromatic compounds are readily spread in the environment and pose great potential toxicity concerns. Here, we report the simultaneous degradation of three isomers of mononitrophenol in a single system by employing a consortium of three bacteria, both in flasks and lab-scale sequential batch reactors. The results demonstrate that simultaneous biodegradation of three mononitrophenol isomers can be achieved by a tailor-made microbial consortium immobilized in sequential batch reactors, providing a pilot study for a novel approach for the bioremediation of mixed pollutants, especially isomers present in wastewater.

Efficient induction of immune tolerance to coagulation factor IX following direct intramuscular gene transfer.

BACKGROUND: The formation of inhibitory anti-factor IX (anti-FIX) antibodies is a major complication of FIX protein replacement-based treatment for hemophilia B. It is difficult to treat patients with anti-FIX antibodies. Gene therapy is emerging as a potentially effective treatment for hemophilia. Direct i.m. injection of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy. However, the development of anti-FIX antibodies following i.m. of AAV may impede its application to patients. OBJECTIVE: We aimed to investigate induction of immune tolerance to human FIX (hFIX) by i.m. of AAV1, further validating i.m. of AAV1 for hemophilia B gene therapy. METHODS AND RESULTS: Cohorts of hemostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received i.m. of AAV-hFIX. Human FIX antigen and anti-hFIX antibodies were examined. I.m. of 1 x 10(11) vector genomes (VG) of AAV2 elicits formation of anti-hFIX antibodies comparable to those by hFIX protein replacement. I.m. of 1 x 10(11) VG of AAV1 results in expression of therapeutic levels of hFIX (up to 950 ng mL(-1), mean = 772 ng mL(-1), SEM +/- 35.7) and hFIX-specific immune tolerance in C57BL/6 mice. CONCLUSIONS: A single i.m. of AAV1 can result in efficient expression of therapeutic levels of hFIX and induction of hFIX tolerance in hemostatically normal and hemophilic B mice. Our results substantiate the prospect of i.m. of AAV1 for hemophilia B gene therapy and FIX tolerance induction.

Phase II study of weekly vinorelbine and 24-h infusion of high-dose 5-fluorouracil plus leucovorin as first-line treatment of advanced breast cancer.

We prospectively investigated the efficacy and safety of combining weekly vinorelbine (VNB) with weekly 24-h infusion of high-dose 5-fluorouracil (5-FU) and leucovorin (LV) in the treatment of patients with advanced breast cancer (ABC). Vinorelbine 25 mg m(-2) 30-min intravenous infusion, and high-dose 5-FU 2600 mg m(-2) plus LV 300 mg m(-2) 24-h intravenous infusion (HDFL regimen) were given on days 1 and 8 every 3 weeks. Between June 1999 and April 2003, 40 patients with histologically confirmed recurrent or metastatic breast cancer were enrolled with a median age of 49 years (range: 36-68). A total of 25 patients had recurrent ABC, and 15 patients had primary metastatic diseases. The overall response rate for the intent-to-treat group was 70.0% (95% CI: 54-84%) with eight complete responses and 20 partial responses. All 40 patients were evaluated for survival and toxicities. Among a total of 316 cycles of VNB-HDFL given (average: 7.9: range: 4-14 cycles per patient), the main toxicity was Gr3/4 leucopenia and Gr3/4 neutropenia in 57 (18.0%) and 120 (38.0%) cycles, respectively. Gr1/2 infection and Gr1/2 stomatitis were noted in five (1.6%) and 59 (18.7%) cycles, respectively. None of the patients developed Gr3/4 stomatitis or Gr3/4 infection. Gr2/3 and Gr1 hand-foot syndrome was noted in two (5.0%) and 23 (57.5%) patients, respectively. Gr1 sensory neuropathy developed in three patients. The median time to progression was 8.0 months (range: 3-25.5 months), and the median overall survival was 25.0 months with a follow-up of 5.5 to 45+ months. This VNB-HDFL regimen is a highly active yet well-tolerated first-line treatment for ABC.

Discovery and structure-activity relationships of imidazole-containing tetrahydrobenzodiazepine inhibitors of farnesyltransferase.

2,3,4,5-Tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepines were found to be potent inhibitors of farnesyltransferase (FT). A hydrophobic substituent at the 4-position of the benzodiazepine, linked via a hydrogen bond acceptor, was important to enzyme inhibitory activity. An aryl ring at position 7 or a hydrophobic group linked to the 8-position through an amide, carbamate, or urea linkage was also important for potent inhibition. 2,3,4, 5-Tetrahydro-1-(1H-imidazol-4-ylmethyl)-7-(4-pyridinyl)-4-[2-(t rifluo romethoxy)benzoyl]-1H-1,4-benzodiazepine (36), with an FT IC(50) value of 24 nM, produced 85% phenotypic reversion of Ras transformed NIH 3T3 cells at 1.25 microM and had an EC(50) of 160 nM for inhibition of anchorage-independent growth in soft agar of H-Ras transformed Rat-1 cells. Selected analogues demonstrated ip antitumor activity against an ip Rat-1 tumor in mice.

Regulation of mannose 6-phosphate/insulin-like growth factor II receptor distribution by activators and inhibitors of protein kinase C.

The tumor-promotor phorbol dibutyrate (PDBt) increases the binding of a neoglycoprotein containing mannose 6-phosphate (Man6P) and of insulin-like growth factor II (IGF-II) to the Man6P/IGF-II receptor at the cell surface. This effect is dependent on time and concentration and is also seen with synthetic 1-oleoyl-2-acetyl-sn-glycerol, but not with 4 alpha-phorbol, an inactive tumor-promoter. The increase is due to a 3-4-fold increase in the number of cell-surface, receptors, accompanied by a 1.6-fold increase in ligand-binding affinity. The internalization rate of the Man6P/IGF-II receptor is not affected by PDBt, suggesting that the redistribution of these receptors to the cell surface is due to an accelerated externalization rate. The redistribution of Man6P/IGF-II receptors did not impair the sorting of newly synthesized Man6P-containing ligands while uptake of these ligands is 2-4-fold increased. Inactivation or down regulation of protein kinase C decreased the binding of the Man6P-containing neoglycoprotein to 65% of controls. Incubation of cells with Man6P, IGF-I, IGF-II or epidermal growth factor induces a rapid redistribution of Man6P/IGF-II receptors to the plasma membrane [Braulke, T., Tippmer, S., Neher, E. & von Figura, K. (1989) EMBO J. 8, 681-686]. Incubation with PDBt prevented the effect of growth factors but not that of Man6P on receptor redistribution. Inactivation of protein kinase C did not affect the Man6P/IGF-II receptor redistribution induced by Man6P and growth factors. These data suggest that Man6P, growth factors and activation of protein kinase C by phorbol esters and diacylglycerols modulate Man6P/IGF-II receptor cell-surface binding by at least two independent mechanisms, receptor redistribution as well as an increase of binding affinity, which might be involved in regulation of endocytosis of ligands.

Insulin-like growth factors I and II stimulate endocytosis but do not affect sorting of lysosomal enzymes in human fibroblasts.

The mannose 6-phosphate (Man-6-P)/insulin-like growth factor (IGF) II receptor has separate binding sites for Man-6-P and IGF II. It targets newly synthesized lysosomal enzymes from the Golgi to acidic pre-lysosomal organelles and mediates endocytosis of Man-6-P-containing ligands and IGF II. The two classes of ligands, Man-6-P and IGF II, as well as IGF I and the epidermal growth factor, induce in fibroblasts a transient redistribution of the receptor from internal membranes to the cell surface (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686). Here we show that the redistribution induced by IGF I and IGF II is accomplished without affecting the internalization rate of cell surface receptors. The redistribution results in an increased binding of ligands to the Man-6-P- and IGF II-binding sites of the receptor. Furthermore, the uptake of the lysosomal enzyme arylsulfatase A and of a Man-6-P neoglycoprotein is stimulated 2-3-fold by IGF I and IGF II, and this effect persists for at least 6 h. The IGF I- and IGF II-induced receptor redistribution does not affect the targeting of newly synthesized lysosomal enzymes. These results show that important functions of the Man-6-P/IGF II receptor such as binding and internalization of ligands can be up-regulated by the ligands of this receptor and other growth factors such as IGF I through redistribution of the receptor.