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Biomedical subjects

K J Martin

Publications and source records attributed to K J Martin.

At least 73 records · Page 4Linked to original sources
PubMed1991

The interactions of transcription factors and their adaptors, coactivators and accessory proteins.

Consistent with the complexity of the temporally regulated processes that must occur for growth and development of higher eukaryotes, it is now apparent that transcription is regulated by the formation of multicomponent complexes that assemble on the promoters of genes. These complexes can include (in addition to the five or more general transcription factors and RNA polymerase II) DNA-binding proteins, transcriptional activators, coactivators, adaptors and various accessory proteins. The best studied example of a complex that includes a transcriptional adaptor, accessory proteins and a DNA-binding protein is that involving the herpes simplex virus VP16 protein. Evidence suggests that the adenovirus E1a protein and the cellular Sp1 and CTF/NF1 transcription factors also function through adaptors or coactivators. Each additional component of the transcription complex provides the cell with another point at which to exert control of gene expression.

Animals↗
PubMed1991

Androgens potentiate the effects of erythropoietin in the treatment of anemia of end-stage renal disease.

Since androgens may increase the sensitivity of the erythroid progenitors to erythropoietin, the present studies were designed to investigate the effect of administration of androgens on the actions of exogenous erythropoietin (EPO) in hemodialysis patients. Studies were performed in a group of 15 adult male hemodialysis patients. Seven patients were treated with EPO alone at a dose of 2,000 U intravenously (IV) three times a week. An additional group of eight patients was treated with 2,000 U of EPO three times a week and also received 100 mg of nandrolone decanoate intramuscularly (IM) each week. After 12 weeks of therapy, hematocrit values increased slightly in the group receiving EPO alone, from 25.3 +/- 0.8 to 27.4 +/- 1.5. In contrast, EPO in combination with nandrolone decanoate resulted in a greater increase in hematocrit values, from 24.4 +/- 1.4 to 32.9 +/- 1.8 (P less than 0.001). The results show that the groups receiving low-dose EPO alone had a poor erythropoietic response. In contrast, patients receiving androgen in addition to EPO had a significantly greater increase in hematocrit values with treatment. Transfusions were eliminated in both groups of patients. These data show that androgen therapy significantly augments the action of exogenous EPO such that lower doses of EPO are sufficient for an adequate hematopoietic response.

Anabolic Agents↗
PubMed1991

Regulation of cytosolic calcium by parathyroid hormone and oscillations of cytosolic calcium in fibroblasts from normal and pseudohypoparathyroid patients.

UNLABELLED: The second messengers for PTH action on fibroblasts have not been determined. The hypothesis that Ca2+ is a second messenger was examined by spectrofluorometric measurement of cytosolic calcium ([Ca2+]i), of fura-2-loaded human dermal skin fibroblasts. PTH induced: 1) transient elevations of [Ca2+]i due to both Ca2+ influx and intracellular release which were independent of CAMP generation, and 2) membrane depolarization. PTH treatment of fibroblasts depolarized by KCl induced oscillations of [Ca2+]i, but spontaneous oscillatory activity was not observed. Transient elevations of [Ca2+]i similar to that induced by PTH were observed with PTH-related peptide (PTHrp). PTH regulation of [Ca2+]i was normal in fibroblasts from patients with pseudohypoparathyroidism (PHP) type Ia (deficiency of the stimulatory GTP-binding protein, Gs), but PTH induced transient decreases in [Ca2+]i in fibroblasts from patients with PHP type Ib (defective PTH receptor). Both PHP Ia and Ib fibroblasts exhibited: 1) spontaneous [Ca2+]i oscillations, possibly due to voltage gated Ca2+ entry and Ca2+ induced Ca2+ release; and 2) greater sensitivity than normal fibroblasts for release of Ca2+ from intracellular stores when [Ca2+]i was decreased by lowering external Ca2+. CONCLUSIONS: 1) PTH-induced transient elevations in [Ca2+]i in normal fibroblasts result from Ca2+ entry and intracellular release which are independent of CAMP generation; 2) [Ca2+]i homeostasis is altered in PHP fibroblasts, resulting in Ca2+ oscillations; 3) PTH regulation of [Ca2+]i is altered in PHP Ib, but not in PHP Ia fibroblasts.

Calcium↗
PubMed1990

Evidence for interaction of different eukaryotic transcriptional activators with distinct cellular targets.

The adenovirus E1a protein (E1a), a potent transcription activator, contains a transcriptional activating region. Compared with previously described cellular and viral activators, E1a's activating region has unusual structural properties. It seems that E1a's activating region interacts with a cellular target not required for the function of transcriptional activators with 'acidic' activating regions. By contrast, the target of an acidic activating region is required both by acidic activators and by E1a. It is proposed that the cellular target of E1a's activating region is an 'adaptor' that allows E1a to interact with the basic transcriptional machinery.

Adenovirus Early Proteins↗
PubMed1990

Transcriptional activation by the pseudorabies virus immediate early protein.

The pseudorabies virus immediate early (IE) protein is a potent, promiscuous activator of viral and cellular gene transcription. The promiscuous action of IE protein has led to the suggestion that it functions by an unusual mechanism. Here, we show that IE protein has the two essential features of a typical cellular activator: (1) a transcriptional activation region, and (2) a separable region that directs IE protein, or an unrelated activation region, to the vicinity of the promoter. We map the IE protein activation region to 34 amino acids, demonstrate that it is comparable in strength to the strongest known activation region, and show that it is required for the transcriptional activity of the intact IE protein. The 34-amino-acid IE protein activation region is highly acidic. We provide evidence that it uses the same cellular target as an unrelated acidic activator and a different target from that of a nonacidic activator. Our results provide insight into the function of promiscuous eukaryotic transcriptional activators.

Animals↗
PubMed1990

Phosphorus restriction reverses hyperparathyroidism in uremia independent of changes in calcium and calcitriol.

Phosphorus is a well-known modulator of renal 1 alpha-hydroxylase activity. In early and moderate renal failure it is proposed that dietary Pi reduction ameliorates secondary hyperparathyroidism through increased circulating levels of calcitriol (i.e, 1 alpha, 25-dihydroxycholecalciferol). To gain further insight into the mechanisms by which a low-Pi diet ameliorates secondary hyperparathyroidism in advanced renal insufficiency, studies were performed in five dogs before and 6 mo after the induction of uremia by 5/6 nephrectomy. Glomerular filtration rate decreased from 69.0 +/- 2.3 to 10.5 +/- 0.5 ml/min, immunoreactive parathyroid hormone (irPTH) increased from 66.0 +/- 8.8 to 321.0 +/- 46 pg/ml, and calcitriol decreased from 39.0 +/- 10.4 to 27.0 +/- 6.2 pg/ml. Thereafter, dietary Pi was decreased gradually every 2 wk from 0.95% to 0.6, 0.45, and 0.3%, respectively. Dietary Ca was reduced from 1.6 to 0.6% to prevent development of hypercalcemia. Ionized Ca (ICa) decreased from 5.4 +/- 0.04 to 5.2 +/- 0.05 mg/dl (P less than 0.02), and plasma Pi decreased from 6.3 +/- 0.7 to 4.7 +/- 0.2 mg/dl (P less than 0.05). Calcitriol remained low (23.3 +/- 4.7 pg/ml). However, irPTH gradually decreased from 321.0 +/- 46.0 to 94.7 +/- 22.9 pg/ml (P less than 0.005). These studies indicate that a decrease in dietary Pi from 0.95 to 0.3% suppressed irPTH by approximately 70%. Reduction of irPTH was observed in the absence of a concomitant increase in levels of ICa or calcitriol. These studies suggest that reduction in dietary Pi in advanced renal insufficiency improves secondary hyperparathyroidism by a mechanism that is independent of the levels of calcitriol or plasma ICa.

Animals↗
PubMed1990

Inhibitors of protein kinase-C modulate desensitization of the parathyroid hormone receptor-adenylate cyclase system in opossum kidney cells.

The mechanisms involved in mediating desensitization and down-regulation of renal PTH receptors have not been defined. Recent studies indicate that PTH binding promotes not only stimulation of adenylate cyclase and activation of protein kinase-A (PK-A), but also, stimulation of phospholipase-C, leading to activation of PK-C. PK-C has been shown to alter both receptor and adenylate cyclase function in other systems. Therefore, the present studies were conducted to test whether PK-C might play a role in the regulation of the PTH receptor-cyclase system after exposure to PTH. Exposure of confluent cultures of opossum kidney (OK) cells to rat PTH-(1-34) (100 nM) for 6 h resulted in a 48 +/- 8% (n = 5) decrease in stimulation of cAMP accumulation in response to further exposure to PTH. PTH receptor binding, assessed with 125I-[Nle8,Nle21,Tyr34]rat PTH-(1-34)NH2 as radioligand, was decreased to a similar extent. Phorbol ester (4 beta-12,13-didecanoate; 1 microM) treatment of the cells in the absence of PTH caused a 58 +/- 3% decrease in PTH-stimulated cAMP production, but equilibrium PTH receptor binding was not different from the control value. Both 50 microM H-7 and 0.5 microM Staurosporine (inhibitors of PK-C) completely blocked the effects of phorbol ester. Pretreatment with PTH, however, in the presence of H-7 or Staurosporine resulted in a completely normal cAMP response to restimulation with PTH. Thus, two inhibitors of PK-C completely prevented desensitization to PTH. The decrease in equilibrium PTH binding, seen after incubation with PTH alone, was also blunted by the inhibitors of PK-C. These data indicate that activation of PK-C by stimulation with PTH may play a role in the regulation of the PTH receptor-cyclase system in OK cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Cleavage of parathyroid hormone in macrophage endosomes illustrates a novel pathway for intracellular processing of proteins.

Most ligands which are taken up by macrophages are transported to lysosomes where they are degraded to their constituents by a concert of acid hydrolases. This process requires a number of intracellular events which result in the transport of ligands from light density endosomes to the more dense lysosomes. In contrast, our studies have shown that macrophages may process some incoming ligands in endosomes (Diment, S., and Stahl, P. D. (1985) J. Biol. Chem. 260, 15311-15317) and that cathepsin D, an aspartyl protease, is localized in these organelles (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). Using rabbit alveolar macrophages, which can be subjected to subcellular fractionation, we have traced the intracellular transport and processing of bovine parathyroid hormone (PTH-(1-84]. We present evidence that macrophages internalize PTH-(1-84). Once in endosomes the hormone is cleaved to fragments which include a bioactive peptide, PTH-(1-34), and then the fragments are returned to the extracellular medium, without delivery to lysosomes. The entire cycle from initial binding to release of PTH-(1-34) is achieved within 10-15 min, a time period consistent with findings in vivo. Our data provide evidence for a novel route for processing of an endocytosed ligand.

Animals↗
PubMed1989

Separate binding sites for intact PTH 1-84 and synthetic PTH 1-34 in canine kidney.

The present studies examine the characteristics of parathyroid hormone (PTH) receptor binding in canine basolateral renal cortical membranes using iodinated preparations of intact bovine PTH 1-84 and [Nle8,Nle18,Tyr34] bPTH 1-34 amide. A solid phase lactoperoxidase technique was used to iodinate the bPTH 1-84. The PTH 1-34 analog was iodinated using chloramine T. Both radioligands were purified by reverse phase high pressure liquid chromatography (HPLC). Specific binding of 125I PTH 1-84 reached equilibrium at 3 hours whereas binding of the 125I PTH 1-84 analog reached equilibrium at 45 minutes. Excess bPTH 1-84 resulted in complete inhibition of binding of 125I bPTH 1-84, whereas 22 +/- 1.6% of the bound radioligand remained bound in the presence of excess synthetic bPTH 1-34. These data suggested the possibility of a binding site for the carboxy-terminal region of intact PTH, or binding sites selective for intact hormone. Therefore, additional studies were performed with PTH fragments, PTH 28-53, PTH 35-84, and PTH 53-84. In contrast to previous studies in other systems, these fragments did not result in significant displacement of 125I PTH 1-84. Analysis of binding of 125I PTH 1-84 and 125I [Nle8,Nle18,Tyr34] PTH 1-34 amide, using LIGAND, both indicated a single site model with similar affinities. Thus, the data are consistent either with multiple receptors with similar affinities or a second binding site for bPTH 1-84 on the same receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1989

The noncalcemic analogue of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion.

1,25-Dihydroxyvitamin D (1,25-(OH)2D3) directly suppresses the secretion and synthesis of PTH in vivo and in cell culture. This compound has been used to treat secondary hyperparathyroidism associated with renal failure, but in some patients prolonged treatment with 1,25-(OH)2D3 results in hypercalcemia. An analogue of 1,25-(OH)2D3 with little or no calcemic activity, 22-oxacalcitriol (OCT), was recently developed. We confirmed this lack of calcemic activity by acute and chronic administration to normal rats. A single intraperitoneal injection of vehicle (propylene glycol), OCT, or 1,25-(OH)2D3 (1.0 micrograms/rat) increased calcium by 0.32, 0.30, and 1.40 mg/dl, respectively. When rats were given daily injections of vehicle or 0.5 micrograms of either 1,25-(OH)2D3 or OCT for 4 d, calcium did not change in the rats receiving vehicle or OCT, but increased from 8.4 to 11.4 mg/dl in the rats treated with 1,25-(OH)2D3. In primary cultures of bovine parathyroid cells, 10 nM OCT was as active as 10 nM 1,25-(OH)2D3, suppressing PTH release by 33%. This suppression is due, at least in part, to blocking of transcription of the PTH gene. Using a probe prepared by random prime labeling of an Msp I fragment of plasmid PTHm122, we found that a single 40-ng dose of OCT or 1,25-(OH)2D3 depressed PTH mRNA levels by 70-80% by 48 h when compared with vehicle. Thus, OCT is a very effective suppressor of PTH secretion with virtually no calcemic activity. This analogue may be a valuable tool for the treatment of secondary hyperparathyroidism.

Animals↗
PubMed1989

Protein kinase-A and the effects of parathyroid hormone on phosphate uptake in opossum kidney cells.

Current evidence indicates that signal transduction after receptor binding of PTH involves the stimulation of adenylate cyclase as well as stimulation of phosphoinositide metabolism. Recent studies, showing that PTH alters phosphate transport in opossum kidney cells at concentrations which do not increase cAMP production and that activators of protein kinase-C also alter phosphate transport, have led to the suggestion that there is a dual mechanism for the regulation of phosphate transport by PTH, namely, protein kinase-C at physiological levels of PTH and cAMP at higher levels of PTH. The present studies were designed to evaluate the relationship between cAMP-dependent protein kinase (PK-A), a more sensitive indicator of alterations in cAMP metabolism than measurements of total cellular cAMP, and phosphate transport in opossum kidney cells, in response to bovine (b)PTH 1-34 and [Nle8,Nle18,Tyr34]bPTH 3-34 amide. While bPTH 1-34 markedly stimulated cAMP accumulation (half-maximal stimulation between 1 and 10 nM), PTH 3-34 analog did not. Phosphate transport was inhibited in a dose-dependent manner by bPTH 1-34, with half-maximal effect occurring between 0.1 and 1 nM. [Nle8,Nle18,Tyr34]bPTH 3-34 amide also altered phosphate transport, although this peptide was 3 orders of magnitude less potent than bPTH 1-34. PK-A activity increased in response to bPTH 1-34 and correlated closely with the effects of PTH on phosphate transport. [Nle8,Nle18,Tyr34]bPTH 3-34 amide, which did not appear to increase cAMP, also resulted in a significant increase in the activity of PK-A. Studies of inhibition of cAMP accumulation using 2',5'-dideoxyadenosine demonstrated that while this agent markedly inhibited the accumulation of cAMP in response to PTH, the effects of PTH on phosphate transport were not altered. However, in spite of the reduction in cAMP the activation of PK-A was similar to control. These data indicate that the effects of PTH peptides on phosphate transport are more closely related to changes in the activity of PK-A than to levels of total cAMP. Activation of PK-A in response to PTH is demonstrable at the lowest doses of PTH that alter phosphate transport.

Animals↗
PubMed1989

Combined effects of dexamethasone and 1,25-dihydroxyvitamin D3 on parathyroid hormone secretion in cultured bovine parathyroid cells.

The present studies investigate the effects of glucocorticoids on the function of the parathyroid glands using primary cultures of bovine parathyroid cells. Treatment of parathyroid cell cultures with dexamethasone for 48 h caused a dose-dependent stimulation of PTH secretion. The minimal concentration of dexamethasone required for a significant stimulation of PTH secretion was 0.1 nM. The stimulatory effect of dexamethasone on the secretion of PTH was found within 12 h of treatment with 100 nM dexamethasone. The steroids deoxycorticosterone and cortexolone, which do not have glucocorticoid activity were without effect of PTH secretion. Since glucocorticoids may modulate the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in other tissues, additional studies were performed to evaluate the interactions of glucocorticoids and 1,25-(OH)2D3. Addition of 1,25-(OH)2D3 to parathyroid cell cultures for 48 h significantly suppressed PTH secretion. In the presence of dexamethasone, however, 1,25-(OH)2D3 also significantly decreased PTH secretion, although it did not reduce PTH secretion to control levels. The treatment of parathyroid cell cultures with 100 nM dexamethasone did not affect the parathyroid cell content of 1,25-(OH)2D3 receptors. In summary, these studies indicate that glucocorticoids significantly increase the secretion of PTH in vitro. This stimulatory effect can be inhibited by 1,25-(OH)2D3. The parathyroid gland is an additional site of physiological antagonism of glucocorticoids and 1,25-(OH)2D3.

Animals↗
PubMed1989

Influence of pertussis toxin on parathyroid hormone stimulated cyclic AMP production and phosphate transport in opossum kidney cells.

There is evidence that guanine nucleotide-sensitive (G) proteins intervene in the activation of adenylate cyclase by parathyroid hormone (PTH). Furthermore, recent studies suggest that G proteins may be involved in the activation by PTH of phospholipase C, with subsequent elevation of diacylglycerol, inositol trisphosphate, and intracellular calcium. Since G proteins may be involved in both transduction systems postulated to mediate the actions of PTH, the present studies were performed to evaluate the influence of pertussis toxin, which prevents receptor-mediated activation of G proteins, on the effects of PTH in opossum kidney (OK) cells. In OK cell membranes, pertussis toxin catalyzed the adenosine diphosphate (ADP) ribosylation of a protein with a molecular weight of 41 kd on SDS-PAGE. Cholera toxin catalyzed the ribosylation of two proteins of molecular weight 52 and 45 kd. Pretreatment of the cells with pertussis toxin abolished the labelling of this 41 kd protein, confirming the access of the toxin into the cells and the presence of pertussis toxin-sensitive substrates. The ribosylation of the cholera toxin substrates was unaffected by pertussis toxin pretreatment of the cells. Treatment of OK cells with pertussis toxin did not change the basal levels of cyclic AMP, but increased the levels of cyclic AMP in response to bPTH 1-34 from 355 +/- 17 to 449 +/- 20 pmoles cyclic AMP per 5 minutes per culture. These results were consistent with the inactivation of an inhibitory G protein. Furthermore, PTH-stimulated cyclic AMP generation was inhibited by norepinephrine from 362 +/- 10 to 228 +/- 18 pmole cyclic AMP per 5 minutes per culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1988

Effects of some metabolites of pyritinol (Encephabol) on ACH release from brain slices.

The K+ stimulated release of radioactively labelled ACH from cortical slices obtained from rats was increased by some pyridine derivatives that are metabolites of pyritinol. It appears that this effect is not mediated by an interaction with cholinergic receptors but may be related to the lipid solubility of the compounds. It is suggested that an effect of these metabolites on the membrane of nerve endings may be part of the mechanism by which pyritinol treatment increases ACH levels and release in old rats.

Acetylcholine↗
PubMed1988

Alternative DNA loops regulate the arabinose operon in Escherichia coli.

The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind. This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters. These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations. The data lead to the conclusion that two different DNA loops can form in the ara regulatory region. These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other. One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters. Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases. Formation of this second loop precludes the counterproductive formation of the repression loop.

AraC Transcription Factor↗