Simple in vivo bioassay without radioisotopes for recombinant human erythropoietins.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHuEPO) analogues was developed. The assay took four days, normal mice were used and radioactive compounds were not needed. EPO activity was measured by the increased number of some part of reticulocytes which increased specifically and dose-dependently by the injection of rHuEPO. They were considered to be mostly immature reticulocytes and were counted as the residual particles from blood cells after treatment with a hemolysing reagent. These particles could be counted by conventional automated microcell counters. The assay procedure was simple and easy. The sensitivity, reliability and reproducibility of this method were acceptable for routine in vivo bioassay of rHuEPOs. This method was economical, and can be used instead of the existing bioassays for rHuEPOs.     

Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors

We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC₅₀ of 0.15 μg/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount ofDNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time.     

Immuno-cross-reactivity of histidine and dopa decarboxylases

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.     

Induction of a neutral protease from rat intestine by a dietary manipulation

The activity of a neutral protease is increased in soluble fractions from the intestine of rats after a shift from a protein-free to a casein mash and an injection of triiodothyronine. The increased activity is prevented by the administration of cycloheximide. The time curve of the response of the protease activity in intestine is similar to that of liver DNA synthesis after a shift to 50% casein and triiodothyronine.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Histidine decarboxylase activities in mutant mice deficient in mast cells

The histamine contents were very low in the whole bodies of various types of mutant mice (W^v/W^v, W^v/W, W/W), in which the number of mast cells was decreased, but the L-histidine decarboxylase activities in these mutant mice were not much lower than in control wild type mice. These findings suggest the presence of high histidine decarboxylase activity in cells other than mast cells. Histidine decarboxylase in the whole body of mice was difficult to assay, because the enzyme was rapidly destroyed by proteases, but inclusion of a protease inhibitor, such as Leupeptin, Antipain, Chymostatin, or Pepstatin in the assay mixture permitted the accurate assay of histidine decarboxylase in crude extracts.     

Role of hydroxyproline-rich cell wall protein in growth regulation of rice coleoptiles grown on or under water

The elongation of both intact and excised rice coleoptiles waspromoted when they were submerged in water. Amino acid analysisof the cell wall revealed that air-type coleoptiles (grown onthe surface of water) contained more hydroxyproline than water-typeones (grown under water). The suppression of hydroxylation ofpeptidyl proline under water was confirmed with air-type sectionsby examining the imino acid content, ¹⁴C-proline incorporationinto the cell wall and its modification by ,'-dipyridyl. Also,dipyridyl significantly promoted the growth of floated sectionsto the level of submerged sections. Therefore, the lower hydroxyprolinecontent caused by lower oxygen tension in water is concludedto be one of the factors promoting growth of rice coleoptilesunder water. However, the hydroxyproline content in the cellwall decreased with growth of both air-and water-type coleoptiles;thus hydroxyproline-rich cell wall protein can not be regardedas the final growth cessation factor in rice coleoptiles. (Received December 17, 1979; )