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The application of extractants in whole-cell biocatalysis can have a positive impact on industrial fermentations, in terms of productivity, total amount of product produced, and cell growth. When a product is continuously removed from the microorganism surroundings, product inhibition will be diminished. The strategy is exemplified using phenol as the product and the extractant is selected for solvent-impregnated resins, which can prevent emulsification problems that are commonly encountered in in situ extractive recovery of fermentation products. A systematic approach for selection of superior extractants in whole-cell biocatalysis is discussed in this paper. Three criteria are taken into account, namely, extractant toxicity (log P o/w values), product selectivity, and extractant regeneration. ©2008 American Chemical Society.

Bacillus subtilis forms dormant spores upon nutrient depletion. Under favorable environmental conditions, the spore breaks its dormancy and resumes growth in a process called spore germination and outgrowth. To elucidate the physiological processes that occur during the transition of the dormant spore to an actively growing vegetative cell, we studied this process in a time-dependent manner by a combination of microscopy, analysis of extracellular metabolites, and a genome-wide analysis of transcription. The results indicate the presence of abundant levels of late sporulation transcripts in dormant spores. In addition, the results suggest the existence of a complex and well-regulated spore outgrowth program, involving the temporal expression of at least 30% of the B. subtilis genome. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a dose-dependent increase in labelig index (LI) during 12 days of culture. The basal cells were more sensitive to CSC exposure than non-basal cells during the first 6 to 8 culture days. However, in squamous metaplastic foci developing after culture day 6, both basal and non-basal cells in the mid-part of the epithelium were labeled. Physiological concentrations of all-trans retinol stimulated the non-basal LI and inhibited the basal cell LI. Compared with dimethylsulfoxide (DMSO), all retinol concentrations used in the present study inhibited the basal cell LI at each time point examined (4-12 days culture). Exposure of tracheal rings to retinol, either before or after exposure to CSC, or simultaneous exposure to retinol and CSC, clearly decreased the CSC-induced basal cell proliferative activity depending on the retinol concentration used. It is concluded from the present study that squamous metaplasia induced by vitamin A-deficiency or by CSC originates mainly from basal cells and that for the maintenance of these lesions, both basal and non-basal cells play a role. Furthermore, all-trans retinol inhibited CSC-induced basal cell proliferation. Chemicals/CAS: retinol, 68-26-8, 82445-97-4; Dimethyl Sulfoxide, 67-68-5; Vitamin A, 11103-57-4

Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium-tryptone soy broth (TSA)-the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185-209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell. © 2008 Elsevier B.V. All rights reserved. Chemicals / CAS: dipicolinic acid, 17606-33-6, 499-83-2; Picolinic Acids; dipicolinic acid, 499-83-2

Background: Milk products are a potential matrix for fortification with synthetic folic acid or natural 5-methyltetrahydrofolate (5-CH 3-H4folate) to enhance the daily folate intake. In milk, folate occurs bound to folatebinding proteins (FBP). Our previous studies with an in vitro gastrointestinal model showed that 70% of the initial FBP content of the milk product was retained in the duodenal lumen. While folic acid remained bound to FBP after gastric passage, 5-CH3-H4folate was mainly present as free folate in the duodenal lumen. Aim of the study: To investigate the effect of FBP on the absorption of folic acid and 5-CH 3-H4folate from the intestinal lumen. Methods: The transport of [3H]-folic acid and [14C]-5-CH 3-H4folate across enterocytes was studied in the presence or absence of bovine FBP using monolayers of Caco-2 cells grown on semi-permeable inserts in a two-compartment model. The apparent permeability coefficients (Papp) of folic acid and 5-CH3-H 4folate were determined and compared with the permeability of reference compounds for low (mannitol) and high (caffeine) permeability. Results: The transport from the apical to the basolateral side of the Caco-2 cells was higher (P < 0.05) for folic acid (Papp = 1.7*10-6 cm/s) than for 5- CH3-H4folate (Papp = 1.4*10-6 cm/s) after 2 h incubation to 1 μM folic acid or 5-CH3-H4folate test solutions (pH 7). The permeability of folic acid and 5-CH3-H4folate across Caco-2 monolayers appeared to be higher (P < 0.05) than that of mannitol (Papp = 0.5*10-6 cm/s) but lower (P < 0.05) than that of caffeine (Papp = 34*10-6 cm/s). The addition of FBP to the medium led to a lower (P < 0.05) intestinal transport and cellular accumulation of folic acid and 5-CH3-H4folate. Conclusions: Compared to the reference compounds, folic acid and 5-CH 3-H4folate showed a moderate permeability across Caco-2 cells, which indicates that folate absorption from the intestinal lumen is not likely to be complete. The intestinal transport of folic acid and 5-CH 3-H4folate was found to be dependent on the extent of binding to FBP at the luminal side of the cells. © Steinkopff Verlag 2004.

An amphotropic retroviral vector, LgAL(ΔMo + PyF101) containing a human adenosine deaminase (ADA) cDNA was used to optimize procedures for the lasting genetic modification of the hematopoietic system of mice. The highest number of retrovirally infected cells in the hematopoietic tissues of long- term reconstituted mice was observed after transplantation of bone marrow (BM) cells that had been cocultured in the presence of both interleukin-1α (IL-1α) and IL-3. A significantly lower number was detected when IL-1α was omitted from such cocultures. The yield of cells that generate spleen colony- forming cells (CFU-S) in the BM of lethally irradiated recipients (MRA-CFU- S) significantly improved on inclusion of the adherent cell fraction of cocultures in the transplant. Retroviral integration patterns in MRA-CFU-S- derived spleen colonies showed that an MRA-CFU-S can produce many CFU-S during BM regeneration. Expression of hADA was detected in the circulating white blood cells of long-term reconstituted animals, demonstrating that the LgAL(ΔMo + PyF101) vector is capable of directing the sustained expression of hADA, and in approximately 35% of the transduced MRA-CFU-S-derived spleen colonies. These results should facilitate the development of gene therapy protocols for the treatment of severe combined immunodeficiency caused by a lack of functional ADA. Chemicals/CAS: adenosine deaminase, 9026-93-1; Adenosine Deaminase, EC 3.5.4.4; Fluorouracil, 51-21-8; Genetic Vectors; Hematopoietic Cell Growth Factors; Interleukin-1; Interleukin-3; Stem Cell Factor

Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with β(2→6) linkages] from sucrose or raffinose. Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene (lev), encoding a protein (Lev) consisting of 804 amino acids. Lev showed highest similarity with an inulosucrase of L. reuteri 121 [Inu; producing an inulin polymer with β(2→1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (LevΔ773His) that produced the same levan polymer [with only 2-3 % β(2→1→6) branching points] as L. reuteri 121 cells grown on raffinose. The low degree of branching of the L. reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius, having up to 30 % branches. Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro. Lev is strongly dependent on Ca2+ ions for activity. Unique properties of L. reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli, (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 °C, kinetics that are best described by the Hill equation. © 2004 SGM. Chemicals / CAS: amino acid, 65072-01-7; calcium ion, 14127-61-8; fructose, 30237-26-4, 57-48-7, 7660-25-5, 77907-44-9; inulin, 9005-80-5; levan, 50815-13-9, 9013-95-0; levansucrase, 9030-17-5; raffinose, 512-69-6; sucrose, 122880-25-5, 57-50-1; transferase, 9047-61-4; DNA, Bacterial; Fructans; Hexosyltransferases, EC 2.4.1.-; levansucrase, EC 2.4.1.10; Recombinant Proteins. Molecular Sequence Numbers: GENBANK: AF459437, AL162757, CAA05973, L08445, M18954, P11701, Q06447, Q55242, X02730, AF465251;

Objective: Successful implantation and placentation depend on the interaction between the endometrium and the embryo. Angiogenesis is crucial at this time. In this article we investigate the direct influence of the human embryo on in vitro endometrial angiogenesis. Design: In vitro study. Setting: Human endometrial microvascular endothelial cells (hEMVEC) grown on an in vitro angiogenesis model. Intervention(s): Conditioned media (CM) of human embryos were used to stimulate in vitro angiogenesis. Main Outcome Measure(s): In vitro angiogenesis of hEMVEC. Result(s): Conditioned media of human embryos, containing significant amounts of vascular endothelial growth factor (VEGF)-A, as determined by enzyme-linked immunosorbent assay (ELISA), caused an increase in hEMVEC tube formation. This effect was prevented by soluble VEGF receptor 1, which quenches VEGF-A activity. Recombinant EGF alone and leukemia inhibitory factor in combination with VEGF-A stimulated hEMVEC tube formation. None of the other tested recombinant mediators, which have been described as produced by the early embryo/trophoblast (interleukin (IL) 10, transforming growth factor (TGF) β, placental growth factor, hCG, colony-stimulating factor 1, interferon-γ, insulin-like growth factor I and II, IL-6, platelet-derived growth factor, and TGFα), had an effect on tube formation by hEMVEC. Conclusion(s): For the first time, it is shown that the human embryo is able to stimulate in vitro endometrial angiogenesis at the time of implantation, a process that is mediated by VEGF-A. © 2006 American Society for Reproductive Medicine.

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdΔ24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdΔ24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdΔ24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdΔ24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87δEGFR) tumors with AdΔ24TIMP-3 and AdΔ24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdΔ24 did not significantly affect the antitumor efficacy of this oncolytic agent. ©2005 American Association for Cancer Research. Chemicals / CAS: tissue inhibitor of metalloproteinase 3, 145809-21-8, 164781-40-2; Matrix Metalloproteinase 2, EC 3.4.24.24; Tissue Inhibitor of Metalloproteinase-3

Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvilli. Cells were grown in culture flasks and harvested at different stages of differentiation (days post-confluence: -3, 0, 3, 7, 10, 14, and 18). Two-dimensional gel electrophoresis was used to analyse proteome changes. Approximately 1400 protein spots were detected within the Caco-2 proteome, within the pH 4-7 range. Two-dimensional gel electrophoresis allowed for the detection of 18 proteins from which the levels of expression were found to be associated with differentiation. Of these proteins, 11 were identified by means of MALDI-TOF or NANO-ESI-MS/MS mass spectrometry and include liver fatty acid binding protein (FABL), three forms of α-enolase (ENOA), nucleoside diphosphate kinase A (NDKA), cofilin-1 (COF1), translationally controlled tumour protein (TCTP), mitochondrial 60-kDa heat shock protein (CH60), probable protein disulfide isomerase (ER60), creatine kinase B (KCRB), and glutathione S-transferase α (GTA1). Thus, proteomics revealed that the differentiation-related change in phenotype of Caco-2 involves changes in a variety of distinct biochemical pathways. Some of these proteins have not been shown before to be associated with Caco-2 differentiation (ER60; COF1; CH60; NDKA; TCTP and ENOA). Therefore, processes related to protein folding and disulfide bridge formation, cytoskeleton formation and maintenance, nucleotide metabolism, glycolysis as well as tumorigenesis-associated proteins may be involved in Caco-2 differentiation. Changes in the expression of CH60, TCTP, GTA1, NDKA, and FABL have also been reported to be associated with in vivo colon carcinogenesis. These findings illustrate that a combination of proteomics and cell culture is a useful approach to find markers for Caco-2 differentiation, which could contribute to the comprehension of the process of colon carcinogenesis. © 2003 Elsevier B.V. All rights reserved.

This study was conducted to investigate the role of the enzyme cyclooxygenase (COX) and its prostaglandin product PGE2 in n-6 and n-3 polyunsaturated fatty acid (PUFA)-mediated effects on cellular proliferation of two human colorectal carcinoma cell lines. The long chain PUFAs eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (AA; 20:4n-6) both inhibited cell proliferation of Caco-2 cells compared with the long chain fatty acids α-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6). Neither incubation with PGE2 nor reduction in PGE2 synthesis by EPA compared with AA led to differential effects on cell proliferation in Caco-2 cells. This suggests that n-6 and n-3 PUFA-mediated cell proliferation in Caco-2 cells is not regulated via PGE2 levels. AA and EPA had no effect on growth of HT-29 colon cancer cells with a low COX activity. However, stimulation of COX-2 activity by IL-1β resulted in a decrease in cell proliferation and an induction of cytotoxicity by AA as well as by EPA. Both inhibition of the COX pathway by indomethacin as well as inhibition of direct lipid peroxidation by antioxidants such as vitamin E and C diminished the anti-proliferative effects of AA as well as EPA. Also, malondialdehyde, a product of lipid peroxidation and COX-activity was decreased by addition of vitamin E and partially decreased by indomethacin. These data support the hypothesis that growth inhibitory and cytotoxic effects of PUFAs with methylene-interrupted double bonds such as AA and EPA are due to peroxidation products that are generated during lipid peroxidation and COX activity. Chemicals/CAS: 5 (4 fluorophenyl) 1 [(4 methylsulfonyl)phenyl] 3 trifluoromethylpyrazole, 162054-19-5; 5,8,11,14,17 icosapentaenoic acid, 10417-94-4, 1553-41-9; acetylsalicylic acid, 493-53-8, 50-78-2, 53663-74-4, 53664-49-6, 63781-77-1; alpha tocopherol, 1406-18-4, 1406-70-8, 52225-20-4, 58-95-7, 59-02-9; arachidonic acid, 506-32-1, 6610-25-9, 7771-44-0; ascorbic acid, 134-03-2, 15421-15-5, 50-81-7; carbene, 2465-56-7; celecoxib, 169590-42-5; docosahexaenoic acid, 25167-62-8, 32839-18-2; indometacin, 53-86-1, 74252-25-8, 7681-54-1; linoleic acid, 1509-85-9, 2197-37-7, 60-33-3, 822-17-3; malonaldehyde, 542-78-9; prostaglandin E2, 363-24-6; prostaglandin synthase, 39391-18-9, 59763-19-8, 9055-65-6; Antioxidants; Cyclooxygenase Inhibitors; Dinoprostone, 363-24-6; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; Prostaglandin-Endoperoxide Synthases, EC 1.14.99.1