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J Ham

Publications and source records attributed to J Ham.

At least 37 records · Page 2Linked to original sources
PubMed1998

Phosphorylation of c-Jun is necessary for apoptosis induced by survival signal withdrawal in cerebellar granule neurons.

Cerebellar granule neurons die by apoptosis when deprived of survival signals. This death can be blocked by inhibitors of transcription or protein synthesis, suggesting that new gene expression is required. Here we show that c-jun mRNA and protein levels increase rapidly after survival signal withdrawal and that transfection of the neurons with an expression vector for a c-Jun dominant negative mutant protects them against apoptosis. Phosphorylation of serines 63 and 73 in the c-Jun transactivation domain is known to increase c-Jun activity. By using an antibody specific for c-Jun phosphorylated on serine 63, we show that this site is phosphorylated soon after survival signal withdrawal. To determine whether c-Jun phosphorylation is necessary for apoptosis, we have expressed c-Jun phosphorylation site mutants in granule neurons. c-Junasp, a constitutively active c-Jun mutant in which the known and potential serine and threonine phosphoacceptor sites in the transactivation domain have been mutated to aspartic acid, induces apoptosis under all conditions tested. In contrast, c-Junala, which cannot be phosphorylated because the same sites have been mutated to alanine, blocks apoptosis caused by survival signal withdrawal. Finally, we show that cerebellar granule neurons contain high levels of Jun kinase activity and low levels of p38 kinase activity, neither of which increases after survival signal withdrawal. Mitogen-activated protein kinase activity decreases under the same conditions. These results suggest that c-Jun levels and c-Jun phosphorylation may be regulated by novel mechanisms in cerebellar granule neurons.

Animals↗
PubMed1998

Glutamate pathways mediate somatostatin responses to glucose in normal and diabetic rat hypothalamus.

We investigated the role of hypothalamic glutamate receptors in mediating the stimulatory effect of low glucose (< 5 mM) on somatostatin release. We also studied whether alteration in glutamate release might contribute to the reduced hypothalamic somatostatin response to low glucose observed in diabetic (Goto-Kakizaki) rat hypothalami. Hypothalamic somatostatin release in response to incubation with 1 mM D-glucose was inhibited by the ionotropic glutamate receptor antagonists MK801, D-AP5 and DNQX but not by the metabotropic antagonists L-AP3 or MCPG. The release of somatostatin was increased by the ionotropic agonists NMDA, AMPA and kainate but not by metabotropic agonists t-ACPD or L-AP4. Basal and peak glutamate release in response to incubation with 1 mM glucose, were significantly lower from GK hypothalami There were no significant differences in the basal or stimulated release of serine and GABA. These data indicate that ionotropic NMDA/AMPA/kainate receptors and not metabotropic receptors mediate the effects of glucose on rat hypothalamic somatostatin release. Reduced hypothalamic somatostatin release in response to low glucose in diabetic (Goto-Kakizaki) rats may well be secondary, at least in part, to reduced glutamate release.

Amino Acids↗
PubMed1998

Immortalized human pituitary cells express glycoprotein alpha-subunit and thyrotropin beta (TSH beta).

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.

Antigens, Polyomavirus Transforming↗
PubMed1997

Lethal graft-versus-host disease after simultaneous kidney-pancreas transplantation.

BACKGROUND: This case report is the first documentation of the occurrence and potential source of lethal graft-versus-host disease (GVHD) after simultaneous kidney-pancreas transplantation. The patient was a 27-year-old African-American male who received an ABO-compatible, five HLA antigen-mismatched kidney-pancreas transplant from a 17-year-old African-American female donor, who died after childbirth. METHODS: Preoperative crossmatches using lymphocytotoxicity and flow cytometry were negative. The patient received four blood transfusions within 10 days of transplantation. Immunosuppression consisted of OKT3 induction, and then cyclosporine, azathioprine, and corticosteroids. RESULTS: On postoperative day (POD) 9, the patient became febrile, and leukocytopenia and pancytopenia developed. Immunosuppression was reduced and granulocyte colony-stimulating factor was begun. Cultures were negative, interleukin 6 and interleukin 8 levels were elevated, and a cutaneous rash appeared on POD 18. A skin biopsy demonstrated dermatitis with focal epidermal necrosis consistent with GVHD. In an attempt to identify the source of GVHD, variable-number tandem repeat analysis fingerprinting was performed with DNA from donor splenocytes, from the skin biopsy, as well as from the patient's buccal mucosa. The skin biopsy showed a mixed variable-number tandem repeat analysis type containing DNA fragments matching the recipient and donor. Blood donors were excluded as a source because they were serologically different from the organ donor. The patient developed liver abnormalities and died from multiorgan failure on POD 22. CONCLUSIONS: We speculate that carryover of passenger donor lymphocytes within the transplanted organ were responsible for GVHD. Furthermore, donor traits such as sexual mismatching, African-American race, and alloimmune status may be important potential risk factors for GVHD.

Adolescent↗
PubMed1997

GH3 cells expressing constitutively active Gs alpha (Q227L) show enhanced hormone secretion and proliferation.

cAMP levels in GH3gsp cells (Q227L mutation of Gs alpha), in comparison with uninfected GH3 and GH3vt (with vector alone) cells, were two to three fold (P< 0.01) higher (basal), and 10-20 fold (P<0.001) higher (in the presence of isobutyl methylxanthine, (IBMX)). Proliferation of GH3gsp cells after 7 days in culture, as determined by cell number and [(3)H]thymidine incorporation, were up to 25% (respectively P <0.001 and P < 0.02) higher. After chronic (4 days) but not acute (15 min) exposure to forskolin (10 microM) or dibutyryl cAMP (50 microM) all cell types showed a greater than 200% (P < 0.001) increase in [(3)H]thymidine incorporation. Secretion of prolactin and growth hormone by GH3gsp cells were two to four fold (P <0.001) higher than GH3 and GH3vt cells after 4 h and 10-12 fold (P <0.001) higher after 8 h. In conclusion GH3 cells possessing Q227L have a higher proliferation rate and secrete higher levels of prolactin and growth hormone which are associated with higher levels of cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Bax promotes neuronal cell death and is downregulated during the development of the nervous system.

The Bcl-2 and Bcl-x proteins suppress programmed cell death, whereas Bax promotes apoptosis. We investigated the pattern of expression of Bcl-2, Bax and Bcl-x during neuronal differentiation and development. All three proteins were widely expressed in neonatal rats but, in the adult, Bax levels were 20- to 140-fold lower in the cerebral cortex, cerebellum and heart muscle, whereas Bcl-x was not downregulated in any of the tissues examined. In the cerebral cortex and cerebellum, the decrease in Bax levels occurred after the period of developmental cell death. Further, microinjection of a Bax expression vector into cultured sympathetic neurons, which depend on nerve growth factor for survival, induced apoptosis in the presence of survival factor and increased the rate of cell death after nerve growth factor withdrawal. This effect could be blocked by co-injection of an expression vector for Bcl-xL or for the baculovirus p35 protein, an inhibitor of caspases (ICE-like proteases). These results suggest that, during development, the sensitivity of neurons to signals that induce apoptosis may be regulated by modulating Bax levels and that Bax-induced death requires caspase activity.

Aging↗
PubMed1996

AMP is a component of the low molecular weight mitogenic activity present in human pituitary tumours.

The mitogenic activity of extracts of human non-functional pituitary tumours has been studied. Previously we have reported that the tumour extracts could be resolved into both high (> 13,000) and low (< 3,000) molecular weight fractions using Sephadex G-50 chromatography. Mitogenic activity was assayed by looking at trichloroacetic acid precipitable 3H-thymidine incorporation into GH3 cells. We have now purified the major component of the low molecular weight mitogenic fraction using reversed phase HPLC: the material was identified and found to be 5'-adenosine monophosphate (5'-AMP) using electron spray mass spectrometry. The mitogenic activity of 5'-AMP and the purified tumour extract was confirmed as both produced an increase in GH3 cell number after 4 days of treatment. In conclusion our results show that the major component of the low molecular weight mitogenic activity in human non-functional pituitary tumour extracts is 5'-AMP.

Adenosine Monophosphate↗
PubMed1995

The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding.

The E2 transactivator of bovine papillomavirus type-1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co-operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co-operativity with co-expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild-type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre-initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N-terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID.

Adenoviridae↗
PubMed1995

A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death.

Sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis in its absence. We have investigated the pattern of expression of the Jun and Fos family of transcription factors in dying sympathetic neurons using antibodies specific for each family member. When sympathetic neurons are deprived of NGF, the level of c-Jun protein significantly increases, whereas the levels of the other members of the Jun and Fos family remain relatively constant. c-Jun also becomes more phosphorylated, probably on its amino terminal transactivation domain. When microinjected into sympathetic neurons, an expression vector for a c-Jun dominant negative mutant protects them against NGF withdrawal-induced death, indicating that AP-1 activity is essential for neuronal cell death. Furthermore, overexpression of the full-length c-Jun protein is, in itself, sufficient to induce apoptosis in sympathetic neurons.

Animals↗
PubMed1995

Retrograde cardioplegia does not adequately perfuse the right ventricle.

UNLABELLED: Surgeons often rely primarily on retrograde cardioplegia for myocardial protection, because it provides adequate left ventricular perfusion even in the presence of coronary artery disease. Clinically, however, adequate right ventricular perfusion by retrograde delivery has not been demonstrated. Using intraoperative transesophageal echocardiography, we examined retrograde delivery of cardioplegic solutions by contrast echocardiography, which directly assesses myocardial perfusion. In 15 patients (seven having coronary bypass and eight having valve operations), 4 ml of sonicated Isovue medium was injected retrograde via a coronary sinus catheter. Myocardial perfusion was assessed quantitatively by visual inspection and back-ground-subtracted videodensitometric analysis. In five patients undergoing aortic valve replacement, right and left coronary ostial drainage was estimated during retrograde infusion. Before the aortic crossclamp was removed, myocardial oxygen extraction was calculated in all 15 patients by first delivering warm blood cardioplegic solution for 2 minutes in a retrograde fashion and then taking samples from the cardioplegia line and aortic root. This determined the oxygen extraction ratio across the myocardium at the end of retrograde delivery. Warm blood cardioplegic solution was next given antegrade, and 15 seconds later samples were taken from the cardioplegia line and a right ventricular (acute marginal) vein to determine the oxygen extraction ratio across the right ventricle. As assessed by contrast echocardiography, retrograde infusion resulted in almost four times more perfusion to the left ventricular free wall and septum than to the right ventricular free wall (74 +/- 2 versus 69 +/- 2 versus 20 +/- 2, p < 0.05). In those five patients with an aortotomy the right ostial drainage was less than 5 ml/min whereas left ostial drainage was estimated at 80 ml/min during retrograde administration. Oxygen extraction across the myocardium supplied by retrograde infusion was low after 2 minutes. Conversely, when antegrade cardioplegia was started, right ventricular oxygen extraction rose fourfold (42% +/- 5% versus 11% +/- 1%, p < 0.05), demonstrating that retrograde cardioplegia had not adequately perfused the right ventricular myocardium. CONCLUSIONS: 1. Retrograde cardioplegia provides poor right ventricular myocardial perfusion as assessed by contrast echocardiography and coronary ostial drainage. (2) This poor perfusion is inadequate to meet myocardial demands as demonstrated by the high right ventricular oxygen extraction after a prolonged retrograde infusion. (3) Therefore surgeons must not rely solely on retrograde cardioplegia for right ventricular myocardial protection. This concept is especially important if continuous warm blood cardioplegia is used, because myocardial requirements are then higher.

Blood↗
PubMed1994

The glutamate inhibition of carbachol stimulated inositol phosphate production in rat cortical cells is mediated through an ionotropic NMDA receptor.

Carbachol (0.1 mM) stimulated accumulation of inositol monophosphate (IP1) (3-4 fold of basal, P < 0.001) in fetal rat cortical cells is attenuated by glutamate (at 0.1 mM, 40-70% of carbachol alone, P < 0.001). This inhibition by glutamate was reduced by 2-amino-5-phosphonopentanoic acid (AP5), but not by gamma-D-glutamylaminomethyl sulphonic acid (GAMS) [corrected] or 2-amino-3-phosphonopropionic acid (AP3). The metabotropic receptor agonist (1S,3R)-1-aminocyclopentane-1-3-dicarboxylic acid [(1S,3R)-ACPD] (up to 0.1 mM) had no effect upon carbachol stimulated IP1. Staurosporine and quinacrine were unable to prevent the inhibition of carbachol stimulated IP1 by glutamate. These data suggest that the inhibition of carbachol-stimulated IP1, by glutamate in rat cortical cells is mediated through an NMDA ionotropic receptor.

Animals↗
PubMed1994

Cooperativity in vivo between the E2 transactivator and the TATA box binding protein depends on core promoter structure.

The E2 transactivator protein of bovine papillomavirus 1 (BPV-1) can strongly stimulate complex promoters such as that of the herpes simplex virus thymidine kinase gene but does not efficiently activate minimal promoters that only contain E2 binding sites and a TATA box. Here we show that overexpression of the human, but not yeast, TATA box binding protein (TBP) in transfection experiments overcomes this block and enables E2 to activate a minimal TATA box-containing promoter. This suggests that recruitment of the TFIID complex to such promoters is normally a rate limiting step for transcriptional activation by E2 in vivo. In contrast, minimal promoters that contain an initiator element in addition to a TATA box are efficiently activated by E2 on its own and this activation is only moderately enhanced by TBP overexpression. In such E2-responsive promoters the TATA box or initiator can be functionally replaced by SP1 binding sites. Both the initiator binding protein, TFII-I, and SP1 have been found to interact physically with components of the TFIID complex. Since either TBP overexpression or the presence of an initiator or SP1 binding sites can increase activation by E2, it seems likely that the principal role of the E2 activation domain is to affect a step in the formation of the transcription initiation complex that occurs after TFIID has bound to the promoter. Sequential action of transcription factors, such as TFII-I, SP1 and E2, may be one type of mechanism underlying the widely observed phenomenon of transcriptional synergy.

Adenoviridae↗
PubMed1994

Warm blood cardioplegic induction: an underused modality.

Warm blood cardioplegic induction (WBCI) improves recovery of cardiogenic shock hearts by repaying their energy debt before cold ischemic arrest. This study tests the hypothesis that despite the absence of shock, many hearts are energy depleted and would benefit from WBCI. Twenty-five consecutive (nonshock) patients undergoing open heart operations received antegrade WBCI. Simultaneous samples were drawn from the aortic root and coronary sinus 15 seconds and 2 minutes after cardiac arrest. Samples were analyzed and compared to determine the oxygen consumption, oxygen extraction ratio, and glucose uptake across the left ventricular myocardium. There was a positive linear correlation between oxygen and glucose uptake (p < 0.001). By univariate analysis, severe multivessel disease and high Parsonnet (severity) score were predictors (p < 0.05) of increased metabolic uptake during warm induction. In addition, patients requiring urgent operations (unstable angina, left main disease, or congestive heart failure) and those with a history of hypertension (coronary artery bypass grafting) or left ventricular overload (valve patients) had higher consumption of oxygen and glucose (p < 0.05) compared with patients undergoing elective operations or those without a history of hypertension. In conclusion, warm cardioplegic induction in nonshocked hearts results in increased metabolic uptake indicating energy repayment and correlates with severity of underlying myocardial disease. The need for WBCI is especially great in patients with a history of hypertension or left ventricular overload and those requiring an urgent operation, where increased metabolic extraction was still present after 2 minutes. In addition, even for completely elective patients, WBCI may be useful if the patient has severe multivessel disease or a high Parsonnet score.

Adult↗
PubMed1994

The effects of pyroglutamylglutamylprolineamide, a peptide related to thyrotrophin-releasing hormone, on rat anterior pituitary cells in culture.

Pyroglutamylglutamylprolineamide, which was first discovered in mammalian prostate, differs from thyrotrophin-releasing hormone (TRH) by substitution of glutamic acid for histidine at position two of the tripeptide. Recently, the newly discovered peptide has been identified in substantial concentrations in the rat anterior pituitary gland and, in this study, we have investigated the effects of the peptide on rat anterior pituitary cells in culture. GH3 cells were chosen to examine the possible effects of the new peptide, particularly in relation to its effects on the TRH receptor. This cell-type was deficient, in comparison with normal rat pituitary cells, in the new TRH-related peptide and appeared to be an ideal model cell in which to study the effects of pGlu-Glu-ProNH2. TRH (0.01-100 nM) was found to stimulate the secretion of both GH and prolactin from GH3 cells whereas pGlu-Glu-ProNH2 had no effect within the same concentration ranges. In contrast, at micromolar concentrations pGlu-Glu-ProNH2 exhibited intrinsic TRH-like activity causing stimulation of both GH and prolactin release from GH3 cells. Both TRH and pGlu-Glu-ProNH2 appeared to act through the same intracellular signalling mechanism, causing significant increases in intracellular inositol phosphate within the expected concentration ranges. However, pGlu-Glu-ProNH2 (up to 1 mM) displaced neither [3H]TRH nor [3H]MeTRH from membrane-binding sites on GH3 cells, suggesting that the effects of the new peptide were mediated through a second receptor. The physiological relevance of these effects of pGlu-Glu-ProNH2 requires further investigation.

Animals↗
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↗