Similar nuclear factors mediate stimulation of rat thyroglobulin gene transcription by thyrotropin and insulin-like growth factor-I.

Transcription of the thyroglobulin (TG) gene in rat thyroid FRTL-5 cells is stimulated by two hormones, TSH and insulin-like growth factor-I (IGF-I). The effect of TSH is mimicked by cAMP. Promoter regions of the rat TG gene responsible for hormonal action as well as the nuclear regulatory proteins that interact with these regions were characterized. Minimal promoter that responds to both hormones has been found to be up to -171 basepairs from the transcription initiation site. In DNase-I footprinting analysis, nuclear extracts from cells treated with either of these hormones protected the same two major regions within the minimal promoter. Mutations in these two regions abolished basal, TSH-stimulated, as well as IGF-I-stimulated expression of the fused reporter gene chloramphenicol acetyltransferase. DNA mobility shift assay revealed that cAMP and IGF-I induce binding of similar nuclear proteins to these promoter regions. These results suggest that rat TG gene transcription is regulated by the convergent action of two distinct signaling pathways, possibly involving similar DNA-binding nuclear proteins and regulatory sequences of the TG gene promoter.

Cell cycle modulation by hematopoietic growth factors in myelodysplastic syndromes: analysis by three-color flow cytometry.

Investigations of the effects of hematopoietic growth factors (HGFs) on the cell cycle of cells from myelodysplastic syndrome (MDS) have been hampered by technical difficulties. In this study, using a recently established flow cytometric method that enables detailed analysis of the cell cycle (Gzero-, G1-, S-, and G2/M-phases) of target cells in a heterogeneous cell population, we examined the effects of granulocyte colony-stimulating factor (G-CSF) and other HGFs on the cell cycle of CD13-positive cells (blasts and other malignant myelocytic and monocytic cells) in MDS. The cell cycle response to G-CSF (decrease in Gzero-phase cells and increase in S-phase cells) was heterogeneous among MDS cases. When the data for 13 MDS cases and 15 de novo AML cases were compared statistically, the magnitude of cell cycle activation by G-CSF was weaker for the cells from the MDS cases. Stem cell factor, interleukin-3, or a combination of these HGFs with G-CSF reduced the Gzero-phase cell percentage in all examined MDS cases whose cell cycle was unresponsive to G-CSF alone. When cytosine arabinoside was added to cells with or without stimulation by HGFs, the viable G0-phase cell count was reduced in HGF-stimulated cells compared with unstimulated cells in seven of eight cases. The present results suggest that G-CSF-induced cell cycle stimulation of malignant cells can be expected in a fraction of MDS patients and that even in MDS patients whose cells do not respond to G-CSF, employment of other HGFs and their combination with G-CSF is worth consideration. The results also suggest that a well-designed therapy using HGFs and chemotherapeutic drugs may reduce the quiescent (Gzero) cell count in MDS, which is assumed to be responsible for drug resistance derived from cell kinetics.

The deoxyribonucleic acid regions involved in the hormonal regulation of thyroglobulin gene expression.

Transcription of the thyroglobulin (TG) gene is stimulated by TSH via cAMP. We have characterized the sequence elements responsible for the hormone-dependent expression of TG gene in rat thyroid FRTL-5 cells using internal deletion and linker-scanning mutants of the minimal TG promoter (-170 basepairs) fused with the bacterial chloramphenicol acetyltransferase reporter gene. The TG gene is regulated by at least two regions located between -165 and -140 bp (TG-III) and between -95 and -65 bp (TG-I) from the transcription initiation site. The intervening region can be deleted without significant effect on the promoter activity. Either of the two regions alone does not promote hormone-dependent transcription. A DNase footprinting assay showed that TG-I and TG-III are the principal protein-binding sites and that the proteins interacting with these two regions are induced by TSH or cAMP. These results suggest that the hormone-dependent expression of TG gene may be achieved by cooperative interaction of the proteins bound to TG-I and TG-III.

Differential regulation of multiple c-erbA expression by thyrotropin, insulin and insulin-like growth factor I in rat thyroid FRTL-5 cells.

Regulation of multiple c-erbA gene expression was studied in rat thyroid FRTL-5 cells. Two species of erbA alpha (alpha 1 and alpha 2) mRNA and one species of erbA beta (beta 1) mRNA were identified by Northern blot analysis. Withdrawal of thyrotropin (TSH), insulin and serum from the complete medium resulted in an increase in alpha 1 and alpha 2 erbA mRNA levels without altering the level of erbA beta 1 mRNA. Readdition of TSH, N6,2'-O-dibutyryl cAMP or forskolin caused a transient reduction of alpha 1 and alpha 2 mRNA levels (75-90%) at 3-12 h. The alpha 1 and alpha 2 mRNA levels were restored at 24 h. The TSH action was dose-dependent showing the half-maximal effect at around 10(-9) M. Readdition of TSH did not show any effect on beta 1 mRNA level. The action of TSH was not dependent on ongoing protein synthesis but required ongoing transcription. Inhibitors of thyroid hormone biosynthesis, propylthiouracil and methylmercaptoimidazole, did not show any effect on TSH action. Readdition of insulin or insulin-like growth factor I (IGF-I) caused a dose-dependent reduction of alpha 1 and alpha 2 mRNA levels without any effect on beta 1 mRNA level. Their action was slower than TSH and persistent. The actions of insulin and IGF-I were dependent on both ongoing translation and transcription. These results indicate that TSH and insulin/IGF-I reduce levels of c-erbA alpha 1 and alpha 2 mRNA possibly by two distinct mechanisms without altering c-erbA beta 1 mRNA level in FRTL-5 cells.

Opioid and non-opioid binding of beta-endorphin to bovine adrenal medullary membranes.

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.

Low proportion of G0-phase cells during induction chemotherapy correlates with subsequent remission in acute myeloid leukemia.

Kinetic resistance is assumed to be one of the main mechanisms of drug resistance in acute myeloid leukemia (AML), but the relationship between cell cycle status at diagnosis and achievement of complete remission (CR) is controversial. Based on the possibility that the cell cycle data after starting induction chemotherapy are more important than the pretreatment data, we used 3-color flow cytometry to examine the cell cycle (G0, G1, S, and G2/M phases) of AML cells on days 0, 5, and 9 of the first induction chemotherapy in 20 patients. Cell cycle data at these 3 time points were compared in the patients who achieved CR (CR cases) and in the patients who had persistent leukemia (non-CR cases) after the induction chemotherapy. In the CR cases, there was a tendency for the percentages of G0-phase AML cells on days 5 and 9 to be smaller than that on day 0, while the opposite tendency was observed in the non-CR cases. When cell cycle data were compared between the CR and non-CR cases, the percentage of G0-phase AML cells on day 9 differed significantly (CR cases 6.9% +/- 10.9%, non-CR cases 50.1% +/- 38.4%; P = .0024). This significance remained when the patients' AML subtype was taken into consideration. None of the other cell cycle data at each time point or the hematologic parameters, which may be related to CR achievement, showed differences between the CR and non-CR cases. We emphasize the importance of cell cycle analysis after initiating therapy and suggest that such analysis can identify refractory AML subjects. The identified subjects may be candidates for clinical trials of cell cycle modulators.

Coupling of adrenal medullary opioid receptors to islet-activating protein-sensitive GTP-binding proteins.

Possible coupling of bovine adrenal medullary opioid receptors to islet-activating protein (IAP, pertussis toxin)-sensitive GTP-binding proteins was investigated by studying effects of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and IAP treatment of membranes on opioid binding. Gpp(NH)p inhibited [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) binding by increasing the dissociation constant of [3H]DADLE and membranes, and enhanced slightly [3H]diprenorphine binding. IAP treatment of membranes reduced [3H]DADLE binding and abolished almost completely the Gpp(NH)p inhibition of [3H]DADLE binding. Treatment of membranes with IAP and [32P]NAD resulted in radio-labeling of membrane proteins of approximately 39,000 dalton. DADLE inhibited adenylate cyclase activity in rat brain caudate nucleus. However, DADLE, beta-endorphin, levorphanol and dynorphin A(1-13) did not show any significant inhibitory action on bovine adrenal medullary adenylate cyclase activity. These results suggest that bovine adrenal medullary opioid (DADLE) receptors are linked to IAP-sensitive GTP-binding proteins which are not directly coupled to adenylate cyclase.

Hypokalemic periodic paralysis associated with hypophosphatemia in a patient with hyperinsulinemia.

A 34-year-old man was admitted to the hospital because of acute quadriplegia. On admission, serum potassium was 2.1 mEq/L and serum inorganic phosphate was 1.4 mg/dL. Thyroid function was normal. Serum levels of aldosterone, cortisol, and intact parathyroid hormone were normal. Fasting plasma glucose was 109 mg/dL, and fasting serum insulin was 25.0 U/mL. Shortly after intravenous supplementation of potassium, muscle strength was normalized. Oral glucose tolerance test revealed impaired glucose tolerance and hyperresponse of insulin. During the oral glucose tolerance test, serum potassium and phosphate decreased significantly. These findings suggest that hyperinsulinemia and insulin-induced transmembrane shift of extracellular potassium and phosphate may have been involved in the abnormalities of serum electrolytes and development of hypokalemic periodic paralysis in the present patient.

Case report: silent thyroiditis developed during alpha-interferon therapy.

Alpha-interferon (IFN-alpha) was used for the treatment of chronic active hepatitis C in a 30-year-old woman who was euthyroid but had low titers of antithyroid antibodies before treatment. Two months after the initiation of IFN-alpha therapy she became thyrotoxic. She had nontender diffuse goiter. A laboratory examination revealed elevated levels of serum free thyroid hormones and a suppressed concentration of serum thyrotropin. Titers of antimicrosomal antibodies increased. The anti-thyrotropin receptor antibody was negative. A 99mTcO- scintigram of the thyroid showed reduced uptake. During the IFN therapy free thyroid-hormone levels started to decline. The IFN-alpha therapy was completed 1 month after the onset of thyrotoxicosis. Two months after the completion of the therapy the patient became euthyroid and 99mTcO- uptake was normalized. It is likely that preexisting chronic thyroiditis was exacerbated to cause silent thyroiditis during IFN-alpha therapy. None of the other 11 patients with chronic hepatitis C who had had no anti-thyroid antibodies and were treated with IFN-alpha showed anti-thyroid antibodies and thyroid dysfunction after the therapy. It is advisable to assess anti-thyroid antibodies and thyroid function in patients who are going to receive IFN-alpha treatment.

Spontaneous healing of pancreatic abscess after fistulization to the duodenal bulb.

A 70-year-old man was admitted to the hospital because of sudden, upper abdominal and back pain. Laboratory and image data indicated acute pancreatitis. Shortly after the admission, pancreatic and liver abscess with bacteremia developed. Antibiotic therapy seemed effective. A month later, spontaneous fistulization of the pancreatic abscess to the duodenal bulb was found by gastroduodenal fiberscopy. Injection of contrast medium into the duodenal orifice showed that the fistula was draining the abscess and that no other fistula formed from the abscess. Endoscopic retrograde cholangiopancreatogram indicated no fistula formation to the pancreatic duct. The pancreatic abscess became smaller and was not visible using computerized tomography and ultrasonography 3 months later and thereafter. Closure of the duodenal orifice was ascertained by the endoscopy. It is suggested that retrograde infection from the fistula was prevented by the single fistulization to the acidic duodenal bulb, which is not supposed to allow most bacterial growth. Pancreatic abscess usually necessitates operative treatment, even with fistulization to the alimentary tract. It seems likely that the single, small fistulization to the bulb, in addition to the lack of underlying disease and medical and nutritional support, facilitated the spontaneous healing process.

Possible gliclazide-induced water retention with azotemia.

An 80-year-old woman with diabetes mellitus was treated with gliclazide. Prior to the gliclazide administration, her urinary excretion of albumin, serum urea nitrogen and serum creatinine were normal. After the medication, oliguria, edema and azotemia developed. On the twenty-fourth day when the edema was severe and generalized, gliclazide administration was terminated. On the following day urinary volume increased suddenly (5,740 ml/day). Polyuria persisted for five days. Edema improved and urea nitrogen and creatinine were normalized thereafter. Though the mechanism is not known, the clinical course suggests that gliclazide is the principal causative factor in the water retention and azotemia in this patient.

Renal refractoriness to phosphaturic action of parathyroid hormone in a patient with hypomagnesemia.

A 50-year-old tetanic woman with hypomagnesemia is described. She had partial resection of the stomach and the jejunum at the age of 20 years. Lack of parathyroid hormone (PTH) function was indicated by hypocalcemia, hyperphosphatemia and high tubular reabsorption of phosphate. However, both plasma concentration of PTH and nephrogenous cAMP were normal. Administration of magnesium sulfate completely normalized serum phosphate and tubular transport of phosphate with only a modest increase in nephrogenous cAMP. The present findings suggest that phosphaturic action of PTH is impaired in magnesium deficiency and that steps distal to cAMP production may be responsible for the renal refractoriness to the hormonal action.

Inhibition of adenylate cyclase by GTP and its modulation by opiate receptor in rat caudate nucleus.

Morphine inhibited the adenylate cyclase activity of the crude synaptosomal fraction of the rat caudate nucleus in the presence of BTP, GDP, Gpp(NH)p or ITP. The purine nucleotides themselves had an inhibitory action on the enzyme. Beta-endorphin and Met-enkephalin also inhibited the enzyme in the presence of GTP. The GTP-dependent in inhibitory action of morphine was blocked by naloxone. Various opiates and opioid peptides inhibited the enzyme by up to approximately 20 per cent in the presence of GTP. The relative potency was in higher order of levorphanol greater than beta-endorphin greater than Met-enkephalin greater than morphine greater than pentazocine. Levorphanol was about 50,000 times as potent as its biologically inactive enantiomer, dextrorphan. Morphine enhanced the inhibitory actions of GTP and GTPase-resistant Gpp(NH)p on the adenylate cyclase activity. These results suggest that GTP plays an important role in the regulation of adenylate cyclase activity in the rat caudate nucleus and that the occupation of opiate receptor by agonists inhibits the enzyme through an actual increase in the inhibitory action of GTP, rather than a suppression of the enzymatic degradation of GTP.

Elimination of GTP biphasic regulation of synaptosomal adenylate cyclase by manganese and solubilization.

Effects of divalent cations and solubilization with Lubrol-PX were studied on guanine nucleotide regulation of synaptosomal adenylate cyclase activity of the rat caudate nucleus. In the presence of Mg2+, both GTP and Gpp(NH)p exerted biphasic actions on the membrane-bound adenylate cyclase activity. The K0.5 value for the GTP stimulation of the cyclase was 47 nM, and the value for the GTP inhibition was 4.5 microM. One hundred microM dopamine selectively enhanced the stimulatory phase of the GTP action, whereas 10 microM morphine selectively enhanced the inhibitory phase of the GTP action. When Mg2+ was replaced by Mn2+, the inhibition of the membrane-bound adenylate cyclase by these nucleotides and morphine was completely abolished; but the catalytic activity of adenylate cyclase was not impaired. These results suggest that the inhibitory action of GTP is responsible for the morphine inhibition of synaptosomal adenylate cyclase. Lubrol-solubilized adenylate cyclase prefered Mn2+ to Mg2+ for its activity. The stimulation of adenylate cyclase by either GTP or Gpp (NH)p was eliminated in the Sepharose 6B-fractionated solubilized preparation in the presence of either Mg2+ or Mn2+. Ten mM NaF also failed to activate the fractionated adenylate cyclase. In the fractionated solubilized preparation, GTP and Gpp(NH)p failed to inhibit adenylate cyclase. These results indicate that GTP and Gpp(NH)p are unable to inhibit the resolved catalytic unit of the synaptosomal adenylate cyclase.

Microcomputer-based nonlinear regression analysis of ligand-binding data: application of Akaike's information criterion.

Akaike's information criterion (AIC) (Akaike, H., IEEE Trans. Automat. Contr. AC-19, 716-723 (1974)) was applied to estimate statistically the number of classes of binding sites from ligand-binding data. Several sets of data were analyzed by both the AIC method and the F-test method. Good agreement was obtained between results from both methods. The present results suggest that the AIC method can be a good alternative to the F-test to estimate the number of classes of sites.

A case of total thyroxine-binding globulin deficiency with Graves' disease: fluctuations of plasma triiodothyronine/thyroxine ratio.

A 37-year-old male with total thyroxine-binding globulin (TBG) deficiency associated with Graves' disease is described. Both TBG immunoreactivity and TBG capacity were not detectable in his serum. Serum concentrations of thyroxine-binding prealbumin and albumin were normal. He was initially hyperthyroid. During methimazole-treatment he was maintained in an euthyroid state except for two short hypothyroid periods. His plasma triiodothyronine/thyroxine (T3/T4) ratios during both the untreated hyperthyroid and the methimazole-induced hypothyroid states were higher than those during his methimazole-induced euthyroid state. These findings on changes in his T3/T4 ratio accompanying thyroidal dysfunction were qualitatively comparable with those in patients with Graves' disease with normal TBG levels: that both untreated hyperthyroid and methimazole-induced hypothyroid patients showed higher T3/T4 ratios than methimazole-induced euthyroid patients. These results may provide indirect evidence that changes in hormonal secretion and conversion that raise T3/T4 ratio can occur in thyroidal dysfunctions even in the complete absence of TBG.

Solubilization of adrenal medullary opioid receptors.

The opioid-binding activity of digitonin extract of bovine adrenal medullary membranes was studied. [3H]Diprenorphine binding to the solubilized material was rapid and saturable. The dissociation constant of [3H]diprenorphine binding was 0.76 nM. Several opioids displaced the [3H]diprenorphine binding. The complex of [3H]diprenorphine and the solubilized binding sites was eluted as a single peak on a Sepharose 6B column and showed an apparent molecular weight of 200,000. These results indicate that active opioid receptors are solubilized with digitonin from bovine adrenal medullary membranes.