Ontogeny of intestinal safety factors: lactase capacities and lactose loads.
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We measured intestinal safety factors (ratio of a physiological capacity to the load on it) for lactose digestion in developing rat pups. Specifically, we assessed the quantitative relationships between lactose load and the series capacities of lactase and the Na+-glucose cotransporter (SGLT-1). Both capacities increased significantly with age in suckling pups as a result of increasing intestinal mass and maintenance of mass-specific activities. The youngest pups examined (5 days) had surprisingly high safety factors of 8-13 for both lactase and SGLT-1, possibly because milk contains lactase substrates other than lactose; it also, however, suggests that their intestinal capacities were being prepared to meet future demands rather than just current ones. By day 10 (and also at day 15), increased lactose loads resulted in lower safety factors of 4-6, values more typical of adult intestines. The safety factor of SGLT-1 in day 30 (weanling) and day 100 (adult) rats was only approximately 1.0. This was initially unexpected, because most adult intestines maintain a modest reserve capacity beyond nutrient load values, but postweaning rats appear to use hindgut fermentation, assessed by gut morphology and hydrogen production assays, as a built-in reserve capacity. The series capacities of lactase and SGLT-1 varied in concert with each other over ontogeny and as lactose load was manipulated by experimental variation in litter size. (+info)
Disease-inducible transgene expression from a recombinant adeno-associated virus vector in a rat arthritis model.
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Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 1% of the world's population, with significant morbidity and mortality. In this study, we investigated a recombinant adeno-associated virus (rAAV) vector for its potential application in RA gene therapy. rAAV encoding Escherichia coli beta-galactosidase was injected into rat joints which had already been induced into acute arthritis after local lipopolysaccharide (LPS) administration, and the efficiency of in vivo transduction was evaluated. We observed a striking correlation between vector transgene expression and disease severity in arthritic joints. The inflammatory reaction peaked at 3 to 7 days after LPS treatment, and, at the same time, 95% of the synoviocytes had high-level transgene expression. Gene expression diminished to the basal level (5%) when the inflammation subsided at 30 days after LPS treatment. More importantly, the diminished transgene expression could be efficiently reactivated by a repeated insult. The transgene expression in normal joints transduced with rAAV remained low for a long period of time (30 days) but could still be induced to high levels (95%) at 3 to 7 days after LPS treatment. This is the first demonstration of disease state-regulated transgene expression. These findings strongly support the feasibility of therapeutic as well as preventative gene transfer approaches for RA with rAAV vectors containing therapeutic genes, which are expected to respond primarily to the disease state of the target tissue. (+info)
Transcription of the pcbAB, pcbC and penDE genes of Penicillium chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs.
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Glucose repressed transcription of the penicillin biosynthesis genes pcbAB, pcbC and penDE when added at inoculation time to cultures of Penicillium chrysogenum AS-P-78 but it had little repressive effect when added at 12 h and no effect when added at 24 or 36 h. A slight increase in the expression of pcbC and penDE (and to a smaller extent of pcbAB) was observed in glucose-grown cultures at pH 6.8, 7.4 and 8.0 as compared with pH 6.2, but alkaline pHs did not override the strong repression exerted by glucose. Transcription of the actin gene used as control was not significantly affected by glucose or alkaline pHs. Repression by glucose of the three penicillin biosynthetic genes was also observed using the lacZ reporter gene coupled to each of the three promoters in monocopy transformants with the constructions integrated at the pyrG locus. Glucose repression of the three genes encoding enzymes of penicillin biosynthesis therefore appears to be exerted by a regulatory mechanism independent from pH regulation. (+info)
Regulation of Rb and E2F by signal transduction cascades: divergent effects of JNK1 and p38 kinases.
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The E2F transcription factor plays a major role in cell cycle regulation, differentiation and apoptosis, but it is not clear how it is regulated by non-mitogenic signaling cascades. Here we report that two kinases involved in signal transduction have opposite effects on E2F function: the stress-induced kinase JNK1 inhibits E2F1 activity whereas the related p38 kinase reverses Rb-mediated repression of E2F1. JNK1 phosphorylates E2F1 in vitro, and co-transfection of JNK1 reduces the DNA binding activity of E2F1; treatment of cells with TNFalpha had a similar effect. Fas stimulation of Jurkat cells is known to induce p38 kinase and we find a pronounced increase in Rb phosphorylation within 30 min of Fas stimulation. Phosphorylation of Rb correlated with a dissociation of E2F and increased transcriptional activity. The inactivation of Rb by Fas was blocked by SB203580, a p38-specific inhibitor, as well as a dominant-negative p38 construct; cyclin-dependent kinase (cdk) inhibitors as well as dominant-negative cdks had no effect. These results suggest that Fas-mediated inactivation of Rb is mediated via the p38 kinase, independent of cdks. The Rb/E2F-mediated cell cycle regulatory pathway appears to be a normal target for non-mitogenic signaling cascades and could be involved in mediating the cellular effects of such signals. (+info)
A transfection compound series based on a versatile Tris linkage.
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The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity. (+info)
Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae.
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The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription. To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V. cholerae carrying a chromosomally integrated toxT::lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl beta-D galactoside (X-gal). Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH-ubiquinone oxidoreductase (NQR). In V. alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation. Inhibition of NQR enzyme function in V. cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT::lacZ activity. Increased toxT::lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H. However, the ability of the TcpP/H proteins to activate the toxT::lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium. The flagellar motor of V. cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT::lacZ expression. Thus, the regulation of the main virulence factors of V. cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way. (+info)
Preclinical development of human granulocyte-macrophage colony-stimulating factor-transfected melanoma cell vaccine using established canine cell lines and normal dogs.
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Tumor vaccines and gene therapy have received significant attention as means of increasing cellular and humoral immune responses to cancer. We conducted a pilot study of seven research dogs to determine whether intradermal injection of canine tumor cells transfected via the Accell particle-mediated gene transfer device with the cDNA for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) would generate biologically relevant levels of protein and result in demonstrable histological changes at sites of vaccination. Tumor cell vaccines of 10(7) irradiated canine melanoma cells were nontoxic, safe, and well tolerated. No significant alterations in blood chemistry values or hematological profiles were detected. A histological review of control vaccine sites revealed inflammatory responses predominated by eosinophils, whereas vaccine sites with hGM-CSF-transfected tumor cells had an influx of neutrophils and macrophages. Enzyme-linked immunosorbent assays of skin biopsies from vaccine sites had local hGM-CSF production (8.68-16.82 ng/site of injection) at 24 hours after injection and detectable levels (0.014-0.081 ng/site) for < or =2 weeks following vaccination. Flow cytometric analysis of hGM-CSF-transfected cells demonstrated < or =25% transfection efficiency, and hGM-CSF levels obtained during time-course assays demonstrated biologically relevant levels for both irradiated and nonirradiated samples. These data demonstrate the in vivo biological activity of irradiated hGM-CSF-transfected canine tumor cells and help provide evidence for a valid translational research model of spontaneous tumors. (+info)
Delivery of adenoviral vectors to the prostate for gene therapy.
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Prostate cancer has become the most frequently occurring cancer and the second leading cause of cancer deaths in men. One novel approach to combat prostate cancer is gene therapy. A replication-deficient recombinant adenoviral vector (AdRSVlacZ) expressing bacterial beta-galactosidase (beta-gal) (lacZ) under the control of the Rous sarcoma virus promoter was used to determine which delivery route was best for the transduction of adenoviral vectors to the prostate. Using a canine model, adenoviral vectors were administered by intravenous, intra-arterial, and intraprostatic (i.p.) injections. After injections, the expression of the lacZ gene was measured in canine prostates as well as in various other organs to determine the distribution of the disseminated adenoviral vector by (a) the percentage of cells expressing lacZ in situ (5-bromo-4-chloro-3-indolyl beta-D-galactoside staining), (b) beta-gal enzymatic activity (colorimetric beta-gal assay), and (c) polymerase chain reaction of genomic DNA using primers specific for the adenoviral genome. An i.p. injection of the adenoviral vector resulted in a greater transduction rate and expression level of lacZ in the prostate than either intravenous or intra-arterial (inferior vesical/prostatic artery) injections. Thus, an i.p. (or intratumoral) injection seems to be the best route to treat local regional prostate cancer by viral-based gene therapy. (+info)