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J Ham

Publications and source records attributed to J Ham.

At least 37 records · Page 2Linked to original sources
PubMed1988

Characterization of response elements for androgens, glucocorticoids and progestins in mouse mammary tumour virus.

We have characterized steroid response elements in mouse mammary tumour virus (MMTV) by transient transfection. Four partial inverted repeats of the sequence TGTTCT function as response elements for androgen, as well as for glucocorticoid and progestins, although the relative hormone inductions mediated by each oligonucleotide were different. Mutational analysis of the left half of the palindrome showed that a perfect dyad symmetry is not required for optimum activity as a steroid response element. To investigate potential interactions between steroid receptors and transcription factors we have analysed the minimum sequence requirements for a hormone response. Interestingly, a single 15 bp steroid response element and a TATA box are sufficient for steroid inductions. When the distance between the two elements was increased by up to two turns of the helix the hormone induction initially increased and then gradually declined with no obvious periodicity.

Animals↗
PubMed1988

Evidence that growth hormone depletion and uncoupling of the regulatory protein of adenylate cyclase (Ns) both contribute to the desensitization of growth hormone responses to growth hormone-releasing factor.

Recent data suggest that the response of GH to GH-releasing factor (GRF) is reduced following prior exposure to high concentrations of GRF. However, it is unknown whether this is due to alterations in GRF receptors, adenylate cyclase activity or the size of a GRF-releasable storage pool of GH. In order to clarify these questions we have compared the effects of pretreatment with GRF (10 nmol/l every 2 h for 12 h) with those of pretreatment with somatostatin (SRIF; 1 mumol/l), forskolin (10 mumol/l) and GRF plus SRIF (10 nmol/l and 1 mumol/l added together) on the subsequent responses of GH to GRF (1 pmol/l-10 nmol/l), cholera toxin (10 nmol/l), 3-isobutyl-l-methylxanthine (IBMX) (100 mumol/l) and forskolin (10 mumol/l). Experiments were performed on 4-day monolayer cultures of rat anterior pituitary cells. The cells were pretreated with test substances every 2 h for 12 h and incubated with GRF or forskolin for 3 h. Per cent maximal (Bmax) GH responses to GRF (10 nmol/l) were reduced after pretreatment with both GRF (control, 173% of basal; GRF, 25% of basal; P less than 0.001) and forskolin (98% of basal; P less than 0.001), but were increased after pretreatment with SRIF (246% of basal; P less than 0.02). However, GH responses after pretreatment with GRF plus SRIF were not significantly different from those of the control.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1987

Identification of androgen response elements in mouse mammary tumour virus and the rat prostate C3 gene.

The effect of steroid hormones on mouse mammary tumour virus (MMTV) promoter activity has been analysed in the breast cancer cell lines ZR-75-1 and T-47D 11. Both androgens and progestins were stimulatory in both cell lines, whereas glucocorticoids were stimulatory in only ZR-75 cells. A glucocorticoid response was restored to T-47D cells by the transient expression of a glucocorticoid receptor expression vector, suggesting that endogenous receptors were limiting. The effect of androgen was mediated by a region of MMTV previously shown to be essential for glucocorticoid and progestin responses between nucleotides -201 and -69 upstream of the cap site. Similarly, the effect of androgens on the rat prostate C3(1) gene promoter was analysed in the rat prostate cell line T5. Although these cells had functional androgen receptors which could stimulate MMTV-promoter activity, they were incapable of interacting with androgen response elements to stimulate transcription from the C3(1) promoter.

Androgens↗
PubMed1987

Structural organization and expression of the mouse estrogen receptor.

Complementary DNA clones corresponding to the mouse uterus estrogen receptor mRNA have been isolated and characterized. Nucleotide sequence analysis predicts that full-length cDNA has the potential to code for a polypeptide of 599 amino acids, and comparison with the protein sequences of the rat, human, and chicken estrogen receptors reveals overall homologies of 97%, 88% and 77%, respectively. Genomic clones for the mouse estrogen receptor have been isolated from a cosmid library and used in conjunction with the cDNA clones to study the expression of the receptor in vivo by RNase mapping, primer extension, and Northern blotting. These analyses demonstrate that transcription initiates at multiple sites which span a region of at least 62 base pairs and that the estrogen receptor is encoded by mRNA of approximately 6.5 kilobases in size. There are 10 major starts in total, one of which is situated 31 nucleotides downstream from a TATA box-like motif and coincides with the start of the cDNA clone pMOR8. The ability of the cDNA clone to produce a functional protein was verified by transfection into COS-1 cells which lack endogenous estrogen receptor. The mouse estrogen receptor, in a SV40-based expression vector, was cotransfected with a chimeric marker plasmid consisting of an estrogen response element from the vitellogenin A2 gene linked to the thymidine kinase promoter and the chloramphenicol acetyl transferase gene. In the presence of estradiol chloramphenicol acetyl transferase activity is stimulated by up to 80-fold, while tamoxifen and 4-hydroxytamoxifen act primarily as antiestrogens in this in vitro assay.

Amino Acid Sequence↗
PubMed1986

Chronic stimulation of anterior pituitary cell cultures with CRF leads to the secretion of lipotropin.

Time course studies have shown that the secretion of beta-endorphin-related peptides from primary cultures of rat anterior pituitary reached a plateau after 1 h of stimulation by 10(-7) M corticotropin-releasing factor (CRF); the major peptide released was beta-endorphin 1-31. Further incubation of the cells with CRF led to a decline in the rate of secretion and to the appearance of a different pattern of peptides in which lipotropin was the principal component; analysis of the peptides remaining in the cells, however, showed that beta-endorphin was still the major peptide. Chronic blockade of the anterior pituitary cells with dexamethasone (10(-6) M) resulted in diminished secretion and the released peptides were found to contain a higher proportion of the intact prohormone, pro-opiomelanocortin (POMC). In all the experiments involving stimulation of the pituitary cells with CRF or blockade with corticosteroids it was observed that the pattern of secreted peptides differed from the intracellular patterns, which suggests that selective release can occur from vesicles or cells that contain different segments of POMC.

Animals↗
PubMed1985

beta-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools.

Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of beta-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), beta-lipotropin and 3.5 kDa beta-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of beta-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10(-8) M CRF, the levels of intracellular beta-endorphin and ACTH immunoreactivity were reduced by 15-30% and there was a 4-5 fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa beta-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10(-7) M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10(-6) M dexamethasone the basal secretion of ACTH and beta-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively.

Adrenocorticotropic Hormone↗
PubMed1985

Beta-endorphin processing in pituitary and brain is sensitive to haloperidol stimulation.

Chronic administration of haloperidol (1 mg/Kg) into rats led to a marked increase in the level of beta-endorphin related peptides in the pars intermedia and brain stem. Using a combination of ion-exchange chromatography and radioimmunoassay, the increase in beta-endorphin under haloperidol treatment was confined to the acetylated derivatives: alpha,N-acetyl beta-endorphin 1-26, alpha,N-acetyl beta-endorphin 1-27 and alpha,N-acetyl beta-endorphin 1-31; there was no change in the level of the NH2-peptides. Haloperidol had no effect on the beta-endorphin related peptides in the anterior pituitary or hypothalamus. These findings suggest that haloperidol can influence not only the level of beta-endorphin related peptides in pituitary and brain, but also their posttranslational processing, possibly serving to regulate the level of biologically active beta-endorphin 1-31.

Animals↗
PubMed1985

Cloning of the CDC7 gene of Saccharomyces cerevisiae in association with centromeric DNA.

The cell-division-cycle gene, CDC7, of Saccharomyces cerevisiae has been cloned in a large plasmid (pCM6) containing an insert of about 45 kb of yeast chromosomal DNA in the yeast-Escherichia coli shuttle vector, YRp7. A subclone (pCM39), having a 7-kb insert with a unique BamHI site, was capable of conferring the ts+ phenotype on a cdc7ts- strain of S. cerevisiae. When this insert was placed in the vector Y1p28, and then cut with BamHI and integrated into the ts- strain, genetic analysis showed the CDC7-complementing activity and an associated vector marker to be linked to the TRP1 locus, i.e., to chromosome IV. Analysis of subclones covering the pCM39 insert showed that pCM52, in which a 3.3-kb EcoRI-HpaI fragment with the unique BamHI site has been cloned, possessed the full complementing activity. Although the CDC7 gene lies across the BamHI site, the main part of the gene appears to be in the 2.2-kb BamHI-HpaI segment of the pCM52 insert. A Northern blot analysis showed this region to give rise to a 1.7-kb mRNA which we conclude is the transcript of CDC7. The large insert of pCM6 also contains a 1.5-kb XhoI fragment which several observations suggest is that containing the centromere (CEN4) of chromosome IV to which the CDC7 gene is closely linked. The 45-kb DNA fragment in pCM6 is believed to be an uninterrupted piece of chromosome IV.

Cell Cycle↗
PubMed1979

Simultaneous modeling of pharmacokinetics and pharmacodynamics: application to d-tubocurarine.

We propose a model of drug pharmacodynamic response that when integrated with a pharmacokinetic model allows characterization of the temporal aspects of pharmacodynamics as well as the time-independent sensitivity component. The total model can accommodate extremes of effect. It allows fitting of simultaneous plasma concentration (Cp) and effect data from the initial distribution phase of drug administration, or from any non-equilibrium phase. The model postulates a hypothetical effect compartment, the dynamics of which are adjusted to reflect the temporal dynamics of drug effect. The effect compartment is modeled as an additional compartment linked to the plasma compartment by a first-order process, but whose exponential does not enter into the pharmacokinetic solution for the mass of drug in the body. The hypothetical amount of drug in the effect compartment is then related to the observed effect by the Hill equation, a nonlinear sigmoid form. Nonlinear least-squares data fitting is used for parameter estimation. The model is demonstrated on two different sets of Cp and effect data for the drug d-tubocurarine (dTC). In 7 normal subjects, the (mean +/- SD) rate constant for equilibration of dTC effect (paralysis) and Cp is 0.13 +/- 0.04 min-1 and the (mean +/- SD) steady-state Cp required to produce 50% paralysis is 0.37 +/- 0.05 microgram/ml.

Adult↗
PubMed1979

A technique for the chronic study of the hepatic uptake and excretion of substances in the conscious pig.

The pig is an ideal animal for studies of hepatic metabolism and the handling of drugs by the liver because of the many similarities in liver function to that in man. In this report we describe for the first time, in this animal, methods for the chronic implantation of sampling catheters in the major hepatic vessels and for the construction of an external biliary shunt. This model has many advantages in that it permits in the conscious animal intermittent, simultaneous and precise sampling of the hepatic uptake and clearance of substances and intermittent sampling of bile, without permanent interruption of the enterophepatic circulation. The effectiveness of this preparation has been assessed in 55 pigs. It was demonstrated that the majority of catheters remained patent for at least three days and in many cases for much longer. No significant alteration in liver function could be shown for at least seven days after surgery.

Animals↗
PubMed1978

Pharmacokinetics and pharmacodynamics of d-tubocurarine during hypothermia in the cat.

To determine the effects of hypothermia on the pharmacokinetics and pharmacodynamics of d-tubocurarine (dTc), serum, biliary, and urinary concentrations were determined and twitch tension monitored following intravenous administration of dTc, 0.7 mg/kg, at 39 (n = 5), 34 (n = 5), and 28 C (n = 6) in cats anesthetized with chloralose and urethane. Time from injection of dTc to maximum neuromuscular blockade was prolonged by hypothermia (28 C). Similarly, moderate (28 C) but not mild (34 C) hypothermia delayed recovery from paralysis. The serum half-life was prolonged 76% and the serum clearance rate decreased 60% by hypothermia (28 C). The combined biliary and urinary elimination of dTc was decreased 47% at 28 C compared with 34 and 39 C. The serum concentration of dTc necessary for neuromuscular blockade was less at 39 C (ED50 0.87 microgram/ml) than at 34 or 28 C (ED50 1.13 microgram/ml). It is concluded that, in vivo, hypothermia antagonizes a dTc-induced neuromuscular blockade but decreases the elimination of dTc. At 28 C the net effect is a prolongation of neuromuscular blockade.

Animals↗
PubMed1978

The experimental assessment of techniques of measuring biliary pressure.

A variety of different methods have been used for the measurement of pressures during operations on the biliary tract. However, there are few data on the experimental evaluation of the methods, and there has been no controlled comparative evaluation of the most commonly used techniques. In these experiments, we have compared the manometric technique of Caroli, the syringe barrel method of Daniel and White et alii, and the constant infusion apparatus of Cushieri. The experiments were performed in pigs because of the similarity of their biliary tract to that of man. Resting and opening pressures were recorded, and flow into the duodenum via the ampulla was measured simultaneously. The manometric and syringe barrel techniques gave highly reproducible measurements of resting and opening pressure, and valid measurements of opening pressure. The constant infusion apparatus gave reproducible measurements of resting pressure, but did not measure opening pressure. The manometric technique was shown to measure changes in the resistance of the choledochoduodenal junction in response to morphine and atropine.

Animals↗