Biomedical subjects
Publications and source records attributed to J C Lang.
Regulation of FGF-4 gene expression is controlled both by elements in the promoter and by an enhancer domain located in the untranslated region of the third exon. We have determined that transcription factor NF-Y binds to the FGF-4 promoter. We further show by mutational analysis that binding of NF-Y is essential for FGF-4 enhancer activity but has minimal effect on activity of the FGF-4 promoter alone.
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Genetically engineered monocytes and macrophages may have potential as effector cells for the adoptive immunotherapy of cancer. As a first step, we have transfected the genes encoding either mouse interferon (IFN)-gamma, human interleukin (IL)-6, mouse IL-4, or mouse tumor necrosis factor (TNF)-alpha into the mouse macrophage cell line, J774A.1 cells using retroviral vectors. In vitro activation of J774A.1 cells by gene modification was assessed by morphological changes, proliferative activity was determined by [3H]-TdR uptake, and cytolytic activity was assessed using an 18-hour chromium-51 (51Cr) release assay. In vivo tumoricidal activity was studied by means of local adoptive immunotherapy using intratumoral injection of transfected effector cells. IFN-gamma gene-transfected J774A.1 [J7(IFN-gamma)] cells developed filamentous processes, increased doubling times, and enhanced tumoricidal activity against three tumor cell lines: the TNF-sensitive fibrosarcoma line WEHI 164 and the TNF-alpha-resistant cell lines B16 melanoma and C1300 neuroblastoma. IL-6-, TNF-alpha-, and IL-4-gene-transfected J774A.1 cells also had augmented tumoricidal activity but did not display any changes in morphology or growth. Cytolytic activity was markedly reduced after the addition of anti-TNF-alpha antibodies. Cytolytic J7(IFN-gamma) cells showed upregulated expression of TNF-alpha messenger RNA. After intratumoral injection of J7(IL-4) and J7(IFN-gamma) cell mixtures, 50% of established B16 melanomas were rejected by C57BL/6 mice, thereby demonstrating synergistic killing. Further studies on gene-transfected macrophages should better define their potential usefulness in tumor immunotherapy.
We report the independent isolation of a rearranged FGF-4 gene from a patient with chronic myeloid leukaemia. We show that the FGF-4 gene has been truncated 30 nucleotides 3' to the coding sequence and has been fused to the RNA processing signals from a putative unknown gene on chromosome 15. We demonstrate that the promoter region of the FGF-4 gene is active in NIH3T3 cells and is indeed necessary for transformation. Using the luciferase reporter assay we have shown that the FGF-4 5' flanking sequences possess easily detectable promoter activity in both F9 and HeLa cell lines. 5' deletion analysis of the FGF-4 promoter has delineated regions containing cis-acting elements of functional importance. These regulatory regions are common to both embryonal and somatic cell lines. Electrophoretic mobility shift assay, using nuclear extracts from F9 and HeLa cells, has allowed detection of DNA-protein interactions occurring in the functionally significant regions. Subsequent comparison of the human and murine FGF-4 promoters show that the regions of functional significance are highly conserved. We suggest that the FGF-4 gene may be suppressed through a distal suppressor locus and becomes active when separated from this suppressor.
Down regulation of the c-myc gene is a prerequisite for the differentiation of a number of cell types. Studies have shown that two mechanisms of inactivation are involved in c-myc repression: a block of the elongation of RNA polymerase followed by transcriptional inactivation mediated through promoter sequences. In this study DMS in vivo footprinting was performed on the P2 promoter region of c-myc in differentiated and undifferentiated HL60 cells. A differentiation-specific footprint was observed at G residues immediately upstream of the TATA box. This observation occurred only in cells differentiated for 48 hours or more and hence is likely to be involved in the repression of c-myc by promoter inactivation.
c-MYC was found to repress expression of the c-myc promoter in a transient assay system. Deletion constructs showed the cis-acting element(s) were common to both the P1 and P2 promoters. Further analysis involving replacement of the P2 TATA box and initiator elements with the SV40 late promoter initiator localized the cis-acting elements to the core promoter.
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An experimental method is described for measuring time-release profiles of drugs from various ophthalmic dosage forms. This in vitro method is carried out under conditions nearly representative of those observed in vivo. Specifically, we have reproduced in vitro the small volume and the slow exchange rate of the human precorneal tear reservoir. Volumes on the order of 8-30 microL are achievable. The concentration of drug downstream from the sample reservoir is analyzed nearly continuously and the entire release profiles are appropriate for modeling. This analytical system has been used to study drug release kinetics from controlled release formulations such as gels and suspensions. The method is compared with alternative techniques.
1. Beagle dogs dosed orally with 14C-tazadolene succinate excreted much of the dose in the urine (mean 63.1% in 5 days with most excreted in the first 24 h). A lesser proportion of the dose was excreted in the faeces (mean 20.7%) and again most of this was voided in the first 24 h. 2. Four metabolites were identified and quantified in the urine, namely 3-hydroxy-(M1), 4-hydroxy- (M2a), and 3-methoxy-4-hydroxy-tazadolene (M2b) and N-[2-(phenylmethylene)cyclohexyl]-beta-alanine (M3). 3. In the 24 h urine, M2a and b glucuronides accounted for 17.7% dose, unconjugated M2a and b for 11.3%, and M3 for 18.3%. Insufficient M1 was present to be quantified. The same metabolites were seen in the 24 h faeces, but at lower concn. Thus M2a and b glucuronides, M2a and b, and M3 were 3.2%, 4.9% and 3.5% dose respectively. 4. All three phenols were present in plasma as their glucuronides as well as the beta-alanine derivative. They all had the same tmax of 2 h and t1/2 lambda 1 of the order of 1 h.
1. A quantitative h.p.l.c. plasma assay for oxybutynin (OB) and its active metabolite, N-desethyl oxybutynin (DEOB) is described. The method is linear with coefficients of variation ranging between 4 and 11.8% for OB and 4.6-9.1% for DEOB over the typical concentration range measured. Minimum detectable levels were 0.5 and 5 ng/ml for OB and DEOB respectively from a 2 ml sample. 2. Pharmacokinetic parameters were obtained after a single oral dose of OB and after administration two or three times daily to frail elderly and elderly volunteer groups respectively. Single dose results were also compared with data from young healthy volunteers. 3. There was a wide range in peak blood levels and high levels of parent drug were matched by high DEOB metabolite levels. Plasma levels on repeated administration were as would be predicted from the single dose kinetics. 4. Area under the plasma time course curve for DEOB metabolite was less than or equal to 5 than that of the parent drug. 5. A trend of increasing peak plasma levels and bioavailability was observed with increasing age and frailty, with the differences more apparent between the active elderly and frail elderly groups than between the active elderly and young volunteers. 6. Results indicate that for frail elderly patients a lower initial starting dose of 2.5 mg OB given two or three times a day may provide adequate therapeutic blood levels of the drug.
The problem of optimizing ocular bioavailability of topically applied ophthalmic drugs is discussed. A formula for drug concentration in the tear film is derived using well-known pharmacokinetic relationships and a first-order drug decay model for the tear film. The time integral of the tear film concentration is then related to ocular bioavailability. The results of this analysis show that: (1) high corneal permeability (corresponding to lipophilic compounds) produces the highest bioavailability; (2) the bioavailability of drugs with high corneal permeability is relatively unaffected by drug volume; and (3) by making the dosage volume sufficiently small, a bioavailability improvement factor of approximately 4 can be obtained for drugs with low corneal permeability.
The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.
Expression of P0 RNA in some Burkitt lymphoma cell lines varies independently of levels of RNA derived from P1 and P2. These data suggest the possibility that expression of P0 RNA may be capable of independent regulation. In order to investigate this possibility we have isolated putative regulatory domains flanking P0 RNA starts within the human c-myc gene and analysed both their ability to direct expression of control reporter genes and their ability to interact with specific transcription factors. Regulatory regions necessary for expression of P0 RNA have been located within 131 bp 5' of the first major P0 RNA start. DNAase 1 footprint analysis and gel retardation assays demonstrate binding of transcription factors Sp1, NF1 and CBP to this region. NF1 binds specifically to two consensus sequences. The more distal site overlaps with the binding site for CBP, and it is likely that concomitant binding of NF1 and CBP within the distal region of the P0 promoter is not possible. Previous work from our laboratory has described a negative regulatory domain within the 5' flanking region of c-myc. The P0 promoter resides within this domain and therefore may contain a negative regulator of c-myc gene expression.
Site specific antisera against a synthetic peptide corresponding to the sequence 3-17 of G alpha 12 have been raised and the specificity examined using purified homogeneous Go, Gi2 and Gi containing a 41 kDa alpha-subunit. The distribution of G alpha i2 was investigated in plasma membranes from different tissues and cells and compared to the distribution of G alpha o and other pertussis toxin sensitive G alpha. Considerable amounts of G alpha i2 were found in endocrine tissue especially in membranes from the adrenal and thyroid, in leucocytes and platelets where it constitutes the major, if not only, pertussis toxin-sensitive G alpha, as well as in some cell lines (C6, NG 108-15, S49 cyc-); erythrocytes contained a 41 kDa G alpha i which was different from G alpha i2. G alpha o was present abundantly in nervous tissue, adrenal medulla and cortex but also found in low amounts in other membranes except for lung, liver and blood cells. Subcellular fractionation of cardiac ventricular muscle demonstrated the presence of G alpha o and low amounts of G alpha i2 in sarcolemma, but only 41kDa G alpha i was present in sarcoplasmic reticulum. The importance of the distinct distribution in terms of signal transduction is discussed.
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The involvement of c-myc in the genesis of animal neoplasia is now well documented for several systems. In order to define the precise role played by the myc gene in tumorigenesis, a better understanding of the normal regulation of myc expression is necessary. We have begun a study of the cis-acting regulatory sequences within the 5' flanking domain of the human c-myc gene. Regions important for myc promoter function have been identified by linkage to the coding sequences of the bacterial chloramphenicol acetyltransferase (cat) gene. Promoter deletion studies and in vivo competition assays for c-myc/cat recombinant plasmids have allowed the identification of a proximal 'core' promoter region capable of directing high levels of CAT activity. Further upstream a negative regulatory element (NRE2) has been identified which is capable of repressing cat gene expression and which functions by interaction with a transacting factor(s). Preliminary data suggests detection of NRE2 is dependent on both the type and amount of carrier DNA used in transient CAT assays. Initial experiments further indicate the involvement of at least two other distal regulatory domains, a negative regulatory domain (NRE1) and a putative enhancer-type region (E). In vitro footprint analysis has allowed the identification of DNA binding proteins which interact with NRE2 and the 'core' promoter. NRE2 contains binding sites for transcription factors Sp1 and CTF. The 'core' promoter domain appears to be highly complex and possesses several Sp1 binding sites.
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