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Biomedical subjects

J Ham

Publications and source records attributed to J Ham.

At least 19 recordsLinked to original sources
PubMed1992

How do eukaryotic activator proteins stimulate the rate of transcription by RNA polymerase II?

A large number of activator proteins have now been identified in higher and lower eukaryotes, which bind to the regulatory regions of protein-encoding genes and increase the rate at which they are transcribed by RNA polymerase II. The mechanism by which activators function is being intensively studied and some of the targets of transcriptional activation domains have now been identified. These studies have also revealed novel classes of regulatory factors, which were not anticipated by extrapolating from the principles obtained with prokaryotic promoters.

Animals↗
PubMed1991

Prolonged exposure to N-methyl-D-aspartate increases intracellular and secreted somatostatin in rat cortical cells.

Somatostatin (SRIF) release from fetal rat cortical cells was stimulated by exposure to 10(-5) M N-methyl-D-aspartate (NMDA) (250 +/- 20% of basal at 96 h). A similar but much less potent effect was seen with kainate (KA) but not with quisqualate (Q) which inhibited SRIF release (KA 150 +/- 13%, Q 65 +/- 18% of basal at 96 h). Similar data were obtained for intracellular levels of SRIF. Dose-dependent experiments showed that the EC50 for the stimulatory action of NMDA was 2-3 x 10(-6) M with a Bmax of around 10(-5) M. At 10(-4) M KA and Q but not NMDA reduced tissue content and release of SRIF (KA: 47 +/- 14, 67 +/- 17%; Q: 36 +/- 13, 42 +/- 6% of basal for content and release, respectively). These findings indicate that cortical SRIF content and release is enhanced by exposure to NMDA but not by KA or Q. We suggest that SRIF-containing neurones are sensitive to glutamate damage through the activation of non-NMDA rather than NMDA receptors.

Animals↗
PubMed1991

Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV-1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV-1 E2 expression vector and a series of reporter plasmids containing well-defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co-operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation.

Base Sequence↗
PubMed1991

The papillomavirus E2 protein: a factor with many talents.

The products of the papillomavirus E2 open reading frame play a key role in the regulation of the viral cycle. E2 proteins can activate or repress viral promoters by several distinct mechanisms and viral DNA replication requires the expression of the full-length E2 protein together with the product of the E1 open reading frame. This is an interesting example of how a single eukaryotic DNA-binding protein has evolved to perform several different functions and it provides a valuable model system for studying the regulation of eukaryotic transcription and DNA replication.

Animals↗
PubMed1991

The functional BPV-1 E2 trans-activating protein can act as a repressor by preventing formation of the initiation complex.

The products encoded by the E2 open reading frame of the papillomaviruses are DNA-binding transcription factors involved in the positive or negative regulation of multiple viral promoters. To further understand the mechanisms by which the same transcription factor may act differentially, the full-length BPV-1 E2 protein was expressed and purified from yeast and assayed in vitro for its capacity to modulate transcription. E2 stimulated transcription of the HSV thymidine kinase (TK) promoter when E2-binding sites were positioned in an enhancer configuration approximately 100 bp upstream of the promoter start site. In contrast, the same full-length E2 protein repressed transcription of the HPV-18 E6/E7 P105 promoter. This repression was mediated through binding to the E2 DNA-binding site immediately upstream of the P105 promoter TATA box and could be abrogated by preincubation of the HPV-18 P105 promoter template with the nuclear extract allowing the formation of the preinitiation complex. In vitro DNA-binding experiments with purified E2 and TFIID showed that binding of E2 to its DNA target placed at different positions with respect to the TATA box differentially affects binding of TFIID to its cognate site. In these respects, E2 is similar to the bacteriophage lambda repressor, which can act either as a repressor or an activator of transcription depending on the position of its binding sites relative to the promoter sequences.

Animals↗
PubMed1991

Use of orbital floor steroids in the management of patients with uniocular non-necrotising scleritis.

Most cases of non-necrotising scleritis can be successfully treated with non-steroidal anti-inflammatory drugs. If these are ineffective, then high-dose systemic corticosteroids, with all their attendant side-effects, are usually required. We have used orbital floor injections of depot steroid in the management of nine patients with non-necrotising scleritis in an attempt to avoid the use of systemic steroids, or to allow the dose of steroids to be reduced while maintaining disease control. A temporary reduction in inflammation was achieved in all cases, which allowed the use of systemic steroids to be avoided altogether in two patients and delayed in the others. Non-steroidal anti-inflammatory drugs and systemic corticosteroids remain the mainstay of treatment for non-necrotising scleritis, but orbital floor injections may be a useful adjunct in certain cases.

Adult↗
PubMed1991

Use of cyclosporin in the management of steroid dependent non-necrotising scleritis.

Many patients with non-necrotising scleritis can be treated adequately with non-steroidal anti-inflammatory drugs or steroids. But, as many of them are young, treatment may present problems if they require high doses of steroids to control the scleral inflammation and then relapse when the dosage is reduced. Five such patients were treated with a combination of steroids and low-dose cyclosporin therapy, and in all cases the scleritis remained under control on a much lower dose of steroids than when steroids were used alone. Cyclosporin is therefore a useful drug in the management of younger patients dependent on high-dose steroids to control their scleral inflammation.

Adult↗
PubMed1991

Preliminary characterization of growth factors secreted by human pituitary tumors.

To investigate the secretion of mitogenic factors by human pituitary tumors we have cultured cells from 54 adenomas in serum-free medium. Conditioned media from 28 (52%) elicited dose-dependent stimulation of [3H]thymidine incorporation into rat GH3 cells (22-338% above control), while 14 (26%) inhibited GH3 proliferation. Stimulating activity was observed more frequently in nonfunctional tumor-conditioned medium (73%; n = 22) than in secretory tumor-conditioned medium (37%; n = 32). Of 10 tumour-conditioned media with mitogenic activity for GH3 cells, only 4 produced modest stimulation of HEp2 (human laryngeal carcinoma) cells. In contrast, [3H]thymidine incorporation into A431 (human squamous carcinoma) and PC12 (rat adrenal pheochromocytoma) cells was enhanced by each of 15 tumor-conditioned media (up to 342% and 275%, respectively), 8 of which had shown stimulatory and 2 inhibitory effects on GH3 cells. Gel filtration of pooled conditioned media from 10 nonfunctional tumors showed significant growth-promoting activity for GH3 cells in fractions corresponding to mol wt of 2-3 and 11-18 kDa. Proliferative activity on A431 cells also eluted in two positions; one corresponded to the higher mol wt peak seen with GH3 cells, while the other, not observed with GH3 cells, was in the 3- to 6-kDa range. These findings suggest that cells derived from human pituitary adenoma tissue synthesize and secrete several growth factors, each of which may have its own target cell specificities. These factors have yet to be characterized, but we suggest that they may have a role in stimulating the development or maintenance of human pituitary adenomas.

Adenoma↗
PubMed1991

Kupffer cell-derived cytokines induce the synthesis of a leukocyte chemotactic peptide, interleukin-8, in human hepatoma and primary hepatocyte cultures.

Communication circuits operating between activated monocytes/macrophages and adjacent hepatocytes in the liver effect important alterations in hepatocyte function. We demonstrate here that primary human hepatocytes and hepatoma cells are able to function as effector cells in the recruitment of inflammatory cells in hepatic disease and inflammatory states by synthesizing a neutrophil/lymphocyte chemotactic factor, interleukin-8. We have further investigated the possibility that endogenous factors elaborated by activated peripheral blood monocytes and Kupffer cells in the liver are mediators of hepatocyte-derived interleukin-8 expression. Twenty-four-hour conditioned medium from lipopolysaccharide-stimulated peripheral blood monocytes and nonparenchymal human liver cells enriched for Kupffer cells induced a time-dependent increase in interleukin-8 messenger RNA levels in SK-hepatoma cells over a 24-hr period, similar to that seen for tumor necrosis factor-alpha or interleukin-1 beta induction of interleukin-8 in primary hepatocytes. Exogenously added lipopolysaccharide or recombinant interleukin-6 had no effect. Cell-associated interleukin-8 antigen was present in SK-hepatoma and primary hepatocytes that had been incubated with macrophage-conditioned medium, tumor necrosis factor or interleukin-1 beta. Similarly, neutrophil chemotactic activity was secreted by SK-hepatoma cells, a significant proportion of which could be blocked with interleukin-8--specific antiserum. Preincubation of macrophage-conditioned medium with neutralizing antibodies to tumor necrosis factor-alpha or interleukin-1 beta reduced its interleukin-8 messenger RNA-inducing capacity. Exposure of SK-hepatoma to conditioned medium followed by removal of the stimulus resulted in a rapid down-regulation of interleukin-8 messenger RNA to 50% of the maximum level within the first hour. These data suggest that products derived from activated Kupffer cells can modulate hepatoma cells and primary hepatocyte interleukin-8 gene expression. In addition, macrophage/monocyte-derived tumor necrosis factor-alpha and interleukin-1 beta have major roles in the positive regulatory component of this modulation.

Antigens↗
PubMed1990

The progesterone receptor can regulate transcription in the absence of a functional TATA box element.

We have investigated the importance of the TATA box element in the induction of transcription by the progesterone receptor. Transcription was analyzed from promoters containing a steroid response element upstream of a wild-type or mutated TATA box. Mutation of the TATA box resulted in a loss of correctly initiated transcripts and abolished binding of TATA factor to the TATA box in vitro but did not inhibit transcriptional activation by the progesterone receptor. Thus we conclude that the receptor is able to stimulate the rate of transcription in the absence of a functional TATA box.

Animals↗
PubMed1990

Glycyl-L-glutamine stimulates the accumulation of A12 acetylcholinesterase but not of nicotinic acetylcholine receptors in quail embryonic myotubes by a cyclic AMP-independent mechanism.

Myotubes prepared from the Japanese quail embryo at 9 days gestation were cultivated in the presence of glycyl-L-glutamine (Gly-Gln, beta-endorphin C-terminal dipeptide) or glycyl-glutamic acid (Gly-Glu), and changes in the activity of acetylcholinesterase (AChE) molecular forms and binding of 125I-alpha-bungarotoxin (alpha BGT) to cell surface nicotinic acetylcholine receptors were measured. The A12 oligomer was the major form of AChE in the cultures. The activity of all molecular forms of the enzyme was increased in the presence of Gly-Gln, but Gly-Glu did not alter AChE activity. In cells infected with the temperature-sensitive mutant, La31C, of Rous sarcoma virus (ts-RSV) and transferred to the nonpermissive temperature, the A12 form of AChE was absent, but its activity could be induced following exposure of the cells to Gly-Gln. When cells treated in this way were incubated in the presence of collagenase, there was a small but significant loss of A12 AChE activity, indicating that Gly-Gln stimulated the activity of a pool of this oligomer which was mainly but not entirely intracellular. Neither Gly-Gln nor Gly-Glu influenced 125I-alpha BGT binding after exposure of the cells to the peptides for any duration. Neither Gly-Gln nor Gly-Glu influenced the accumulation of cyclic AMP in the cultures. beta-Endorphin is one of a family of peptides that coexist transiently with acetylcholine in lower motoneurones of vertebrates in the perinatal period. This report provides evidence for the selective trophic activity of one of its derivatives toward the postsynaptic cholinergic system in avian muscle cells.

Acetylcholinesterase↗
PubMed1990

Tumor necrosis factor-alpha, interleukin-8 and chemotactic cytokines.

There is little doubt that the high degree of communication observed during an immune response occurs via sophisticated cell-to-cell mediator circuits. The coordinate expression of specific signals are needed to orchestrate inflammation as the lesion is initiated, maintained, and finally resolved. Each of these phases can be viewed as a unique window of inflammation that is driven by a specific set of mediators. In this manuscript, we have developed the concept that cytokine-cytokine interactions play a prominent role in the elicitation of inflammatory cells. Our data demonstrate that the expression of chemotactic cytokines are both cell and stimulus specific. Interleukin-8, neutrophil chemotactic factor, can be synthesized by a variety of immune and non-immune cells, but the production is dependent upon an appropriate challenge. The production of monocyte chemotactic factor is much more cell restricted and appears to be generated in abundance by only non-immune cells (fibroblasts, endothelial cells, and epithelial cells). These data suggest that the recruitment of cells to a site of inflammation is dependent upon the expression of specific cytokines for both the induction and maintenance of the lesion.

Animals↗
PubMed1989

Transcriptional analysis of the CDC7 protein kinase gene of Saccharomyces cerevisiae.

The 5' flanking region of the CDC7 gene of Saccharomyces cerevisiae has been sequenced to a point 797 nucleotides upstream of the putative translational initiation codon (designated +1). The sequence reveals a number of symmetry elements between -100 to -380 and two blocks of high local AT content between -29 to -75 and -112 to -144. Transcription initiates heterogeneously at about 10 discrete sites up to 110 nucleotides upstream of the putative translational initiation codon. Some minor transcriptional start sites were also observed between the ATG at +1 and a second in-frame ATG at +55, suggesting that CDC7 may also be translationally heterogeneous. Deletion analysis of the CDC7 upstream region has shown that the gene lacks a functional TATA box, and has identified a 30bp element that is necessary for normal CDC7 promoter function during mitosis. This motif has significant homology with a component of the c-fos promoter which acts to regulate c-fos expression by binding a transcription activating factor. The results suggest that the 30bp motif may play a similar role in regulating CDC7 expression and that there may be similarities between factors that regulate CDC7 expression in yeast and c-fos expression in vertebrate cells.

Amino Acid Sequence↗
PubMed1989

Evidence for positive and negative regulatory elements in the 5'-flanking sequence of the mouse sparc (osteonectin) gene.

We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the murine Sparc (osteonectin) gene in parietal endoderm cells and in F9 embryonal carcinoma cells induced to differentiate into parietal endoderm with retinoic acid and cyclic AMP. Varying lengths of flanking sequences extending up to 3.0 kilobase pairs 5' of the transcription initiation site were linked to the bacterial chloramphenicol transacetylase gene in the Bluescript M13- vector. The constructs were tested in transient assays, using a beta-galactosidase plasmid as a transfection control. Sequences between 78 and 169 base pairs upstream of the cap site are the minimum required for cell-type specific promoter activity; this region is dominated by two oligopurine/oligopyrimidine stretches or "GAGA" boxes and is highly conserved between the mouse and bovine genes. Addition of the sequence between -169 and -449, which includes part or all of a third GAGA box, results in increased parietal endoderm specific transcription, up to a maximum of 6.3-fold higher than in undifferentiated F9 cells. Further addition of sequences between -449 and -638 markedly reduces promoter activity in both cell types but parietal endoderm-specific activity is restored in constructs containing 2.2 and 3.0 kilobase pairs of flanking DNA. In addition, we have identified sequences related to the consensus sequence for steroid response elements, one of which is able to confer progesterone-enhanced transcription when tested with a heterologous promoter in steroid responsive cells. These results suggest that negative and positive elements normally interact to regulate the temporal and tissue-specific patterns of Sparc gene transcription seen in vivo.

Animals↗