A novel MPL point mutation resulting in thrombopoietin-independent activation.

Thrombopoietin (TPO) and its receptor (MPL) are important regulators of megakaryopoiesis. MPL belongs to a cytokine receptor superfamily. To date, all constitutively active MPL mutants have been artificially constructed with amino acid substitutions in the transmembrane domain or extracellular domain of the protein, and they activate signal transduction pathways in Ba/F3 cells that can also be activated by the normal MPL. In this paper, we report a novel spontaneously occurring mutation of MPL, with an amino acid substitution of Trp(508) to Ser(508) in the intracellular domain of MPL, that induces the factor-independent growth of Ba/F3 cells. Examination of intracellular signaling pathways demonstrated that the mutant MPL protein constitutively activates three distinct signaling pathways, SHC-Ras-Raf-MAPK/JNK, JAK-STAT, and PI3K-Akt-Bad.

Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: comparison with scintillation proximity assay and streptavidin-coated plate assay.

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC(50) values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low K(m) value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

Effect of alpha 1-adrenoceptor antagonists on urinary bladder function in urethane-anesthetized rats.

1. We investigated the effects of selective alpha 1-adrenoceptor antagonists on rhythmic bladder contraction and cystometrograms as representative of urinary bladder function in urethane-anesthetized rats. 2. The selective alpha 1-adrenoceptor antagonists tamsulosin (0.03-3 micrograms/kg IV), prazosin (0.03-3 micrograms/ kg IV) and bunazosin (0.03-3 micrograms/kg IV) exerted little effect on the amplitude and frequency of rhythmic bladder contraction in anesthetized rats. In contrast, the antipollakiuria agent flavoxate (5 and 10 mg/kg IV) induced a dose-dependent disappearance in frequency without affecting the amplitude of the contractions. 3. Tamsulosin (1 and 3 micrograms/kg IV), prazosin (1 and 3 micrograms/kg IV), and bunazosin (1 and 3 micrograms/kg IV) exerted no effect on the cystometrogram, either. However, flavoxate (5 and 10 mg/kg IV) raised the micturition threshold pressure and prolonged the time to micturition. 4. These results suggest that the alpha 1-adrenoceptor plays little role in urinary bladder contraction in anesthetized rats.

Influence of magnesium deficiency on concentration of calcium in soft tissue of uremic rats.

The influence of magnesium (Mg) deficiency on the concentration of calcium (Ca) in the aorta, heart, and kidney was evaluated in uremic rats. A total of 32 rats were randomly assigned to two groups: one group made uremic by the 5/6 nephrectomy method, and the other serving as sham-operated controls. Both groups were randomly assigned to two subgroups: one group given a Mg-deficient diet and the other fed a Mg-supplemented diet. After 12 weeks on the regimen, all animals were sacrificed. In Mg-supplemented uremic rats, the concentration of Ca in the aorta was higher than in Mg-supplemented control rats. The concentration of Ca in the aorta was further increased in Mg-deficient uremic rats. The concentrations of Ca in the heart and the kidney were also increased in Mg-deficient uremic rats, as compared with Mg-supplemented uremic rats. The concentration of Mg was decreased in the aorta and increased in the kidney of Mg-deficient rats. There was no significant influence of Mg deficiency on the concentration of phosphate in tissue. Results suggest that Mg deficiency in uremia may increase aortic calcification.

Synergistic effect of aurintricarboxylic acid and triflavin in a photochemically induced thrombosis model in rats.

We report here the synergistic antithrombotic effect of aurintricarboxylic acid in combination with a snake venom-derived disintegrin, triflavin, in a photochemically induced thrombosis model in rats. The time to initiation of thrombus was prolonged by i.v. bolus injection of aurintricarboxylic acid at 10 mg/kg. In contrast, time to occlusion was dose-dependently prolonged by both agents, this prolongation being significant with aurintricarboxylic acid at 10 mg/kg i.v. and with triflavin at more than 3 mg/kg i.v. Interestingly, the combination of aurintricarboxylic acid at 3 mg/kg i.v. and triflavin at 1 mg/kg i.v. prolonged not only the initiation of thrombus, but also the time to occlusion.

Decreased contractile effect of endothelin-1 on hyperplastic prostate.

1. The contractile activity, binding activity and localization of endothelin (ET)-1 were evaluated in human nonhyperplastic (control) and hyperplastic prostates. 2. ET-1 caused contraction of both prostates in a dose-dependent manner. However, this contraction was markedly decreased in hyperplastic prostates. 3. Bmax and Kd values of hyperplastic prostates were greater than those of the control. 4. The muscle and proliferative epithelium of hyperplastic prostates showed strong staining for the anti-ET-1 antibody. However, the glandular epithelium of control prostates was weakly stained. 5. These findings indicate that responsiveness to ET-1 is decreased, though the ET-1 and ET-1 receptors increase in the hyperplastic prostate. Namely, the increase in ET-1 receptors is not effective in regulating the contractile response of the prostate, because its expression is rather dominant in proliferated gland. 6. These suggest that ET-1 may not have an important role in the release of the obstructive symptoms of benign prostatic hypertrophy.

Effect of 1,25-dihydroxyvitamin D3 and diltiazem on tissue calcium in uremic rat.

The concentration of calcium was measured in the aorta, heart, and kidney of uremic rats treated with 100 ng/kg/day 1,25-dihydroxyvitamin D3 (1,25 D3) or 60 mg/kg/day diltiazem for 12 weeks. The concentration of calcium was increased in the aorta, heart, and kidney of uremic rats, and was further increased by administration of 1,25 D3. The 1,25 D3-induced increase in calcium in the aorta was inhibited by diltiazem, but this effect was not accompanied by a decrease in serum calcium x phosphate products. Diltiazem had no effect on the 1,25 D3-induced increase of calcium in the heart and kidney. Thus, in uremia 1,25 D3 may promote the calcification of the aorta; calcium antagonists may protect against calcification without a reduction in serum calcium x phosphate products.

High-affinity specific [3H]tamsulosin binding to alpha 1-adrenoceptors in human prostates with benign prostatic hypertrophy.

The binding of a novel radioligand, [3H]tamsulosin, to human prostatic membranes with benign prostatic hypertrophy (BPH) has been characterized. [3H]Tamsulosin rapidly associated with its binding sites in human prostatic membranes with BPH, and the binding reached steady state by 30 min at 25 degrees C. The rate constants for association and dissociation of [3H]tamsulosin binding were calculated to be 0.21 +/- 0.05/nM per minute and 0.01 +/- 0.004/min, respectively. The specific binding of [3H]tamsulosin in human prostatic membranes was saturable and of high affinity (Kd = 0.04 +/- 0.01 nM). The density of [3H]tamsulosin-binding sites (Bmax) was 409 +/- 28 fmol/mg protein. The Kd and Bmax values for [3H]tamsulosin binding in human prostates were significantly lower than those for [3H]prazosin binding. [3H]tamsulosin binding was remarkable for its significantly lower degree of nonspecific binding. Six alpha-adrenoceptor antagonists competed with [3H]tamsulosin for the binding sites in the rank order: tamsulosin > WB4101 > prazosin > S-(+)-isomer > naftopidil > yohimbine. The binding affinities (pKi) of these antagonists for [3H]tamsulosin binding in human prostates closely correlated with their pharmacological potencies (pA2) in prostates. In conclusion, [3H]tamsulosin selectively labels alpha 1-adrenoceptors in human prostates, and thus may become a useful radioligand for the further analysis of these receptors.

Effect of magnesium deficiency on lipid metabolism in uremic rats.

The effects of acute magnesium deficiency on lipid metabolism were examined in five-sixths nephrectomized uremic rats and sham-operated rats. Three weeks after the surgery, both groups were divided into two subgroups. Half of the uremic and sham-operated rats received a magnesium-deficient diet. The rest of the experimental animals received a control diet. After 2 weeks on this regimen, all animals were sacrificed. In uremic rats, magnesium deficiency increased serum triglyceride levels and decreased high-density lipoprotein cholesterol levels as in sham-operated rats. Total serum cholesterol levels were higher in uremic rats than in sham-operated rats with or without magnesium deficiency. Serum free fatty acid levels were increased only in uremic rats with magnesium deficiency. These results suggest that magnesium deficiency worsens several parameters of lipid in uremic rats.

[Studies on small thyroid carcinoma in dialysis patients with hyperparathyroidism].

For twenty long-term hemodialysis patients with medically uncontrolled hyperparathyroidism, subtotal parathyroidectomy (PTX) was performed and histological examination was done for 17 out of them, suspected of thyroid disease from operative findings. Thyroid carcinomas were found in 5 out of 17 patients by biopsied specimens of thyroids. Histological findings of carcinomas were follicular (2 cases) and papillary types (3 cases). In all cases, carcinomas were in occult state and the sizes of carcinomas of 4 cases were small being a diameter less than 10 mm. Among others, findings of follicular adenoma (2 cases) and chronic thyroiditis (1 case) were obtained. The incidence rate of thyroid carcinoma in this report seemed to be rather high as the incidence diagnosed from biopsied specimen at operation. Several factors such as immunological incompetence accompanied by renal failure, metabolic abnormalities of cells induced by parathyroid dysfunction and accelerated aging are considered to be involved as a cause of increased incidence of thyroid carcinoma in hemodialysis patients with hyperparathyroidism.