The role of growth factors in the activity of pharmacological differentiation agents.

Bryostatin-1 inhibits acute myeloid leukemia (AML) in vitroat doses that stimulate the growth of normal hematopoietic progenitors.Although bryostatin-1 has a number of distinct biological activities, those specifically responsible for its antileukemic activity are unclear. We found that bryostatin-1 (10(-8) M) inhibited cell cycling at G(1), induced phenotypic evidence of differentiation, and limited the clonogenic growth of both AML cell lines and patient specimens. This activity was markedly enhanced by granulocyte/macrophage-colony stimulating factor, whereas growth factor-neutralizing antibodies completely inhibited both the differentiating and antileukemic activities of bryostatin-1. Cell cycle inhibition and growth factors were also required for the differentiating activities of two unrelated agents, hydroxyurea and phenylbutyrate. These data suggest that many pharmacological differentiating agents require both cell cycle arrest and lineage-specific growth factors for full activity and may explain why these agents have demonstrated only limited clinical efficacy.     

The biological roles of small GTPases and interacting proteins in plants

Extensive studies on the molecular mechanisms of vesicular trafficking have revealed that molecules involved in this cellular function are remarkably well conserved from yeast to higher plants. However, it is not clear at all how a variety of organisms maintain the individual divergent systems using the common machinery of vesicular traffic. We have been attempting to understand the roles and regulatory mechanisms of vesicular traffic in plants through the study of Rab/Ypt GTPases. Ara proteins are Rab/Ypt homologues ofArabidopsis, which are implicated in the regulation of vesicular traffic. Their biochemical properties are similar to those of the Rab/Ypt proteins from animal and yeast cells. The overexpression ofARA2 orARA4 causes pleiotropic morphological abnormalities in the transgenic tobacco plants. The GTPase cycle of Ara proteins has to be strictly controlled for their proper functions. We have identified two classes of regulator molecules of Ara2 and Ara4. One is the GTPase activating protein (GAP), and the other is the GDP dissociation inhibitor (GDI). GAP has been identified as an activity accelerating the hydrolysis of GTP by Ara2 or Ara4. GDI (AtGDI1) has been isolated as a molecule interacting with Ara4 using a novel method for detecting interactions between foreign molecules in yeast. Further studies on the interacting molecules should unveil the regulatory system of and signal transduction pathway via Ara proteins. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the Internation Prize for Biology “Frontier of Plant Biology”     

Localization of [D-Ala2]deltorphin I-like immunoreactivity in perinatal rat respiratory system

Summary The localization of [D-Ala²]deltorphin I, a -opioid receptor ligand, was studied in the lower respiratory tract of developing rats using an immunohistochemical method. [D-Ala²]-like immunoreactive cells were detected first in the principal bronchus as early as embryonic day 16. As embryos grew, positive cells became gradually visible everywhere from principal bronchi to respiratory bronchioles. The density of positive cells reached the highest level on embryonic day 21, but decreased gradually after birth. Positive cells were no longer seen on postnatal day 30 in any region of the airways. No positive cells were ever found in the trachea or alveoli of rats at any age studied. Ultrastructural examination indicated that the immunoreactive cells possessed a similar morphology to serous or Clara cells of the respiratory epithelium. Immunoreaction products tended to locate at the apical cytoplasm of positive cells. The result suggests that [D-Ala²]-like molecule(s) may be expressed transiently in serous cells or Clara cells, or both, of the rat bronchopulmonary tract. Such a molecule may act as a pulmonary growth-promoting or a differentiation-initiating factor in an early period of lung development.     

Regional differences in glucose transport in the mouse hippocampus

Summary In order to observe glucose transport into the brain, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG), a non-metabolizable and fluorescent glucose analogue, was injected intravenously into mice. After ascertaining that this glucose analogue is non-metabolizable in the brain, the NBDG contents in the blood and brain were measured quantitatively by spectrofluorimetry at 0, 0.5, 2, 5, 10 and 30 min after intravenous injection. The NBDG content in the blood decreased markedly with time, whereas in the brain it rapidly decreased, then gradually increased after 2 min. Glucose transport into the hippocampus was observed with a confocal laser scanning microscope. At 0.5 min, NBGD was seen to be highly concentrated on the vascular wall. Using the confocal mode, it was found that the fluorescence was unevenly distributed on the microvessel wall, suggesting local differences of glucose transport in the vascular wall. At 5 min, the fluoresent intensity of the vascular wall was markedly decreased, whereas relatively intense fluorescence was observed in the cerebral parenchyma of the stratum lacunosum-moleculare and stratum pyramidale of CA3. At 10 min, a weak fluoresence was diffusely distributed in the hippocampus. As to the localization of NBDG in the brain, capillary endothelium (luminal and abluminal membrane), basement membrane, and the feet of the astrocytes are discussed.     

Distribution of an enzyme system producing seaweed flavor: conversion of fatty acids to long-chain aldehydes in seaweeds

The long chain aldehyde-forming enzyme (LCAE) activity that catalyzes formation of long chain aldehydes, such as (8Z, 11Z, 14Z)-heptadecatrienal, (8Z, 11Z)-heptadecadienal, (8Z)-heptadermal, (7Z, 10Z, 13Z)-hexadecatrienal and pentadecanal from linolenic acid, linoleic acid, oleic acid and palmitic acid, in that order, occurs in a wide range of green, brown and red seaweeds. The LCAE activity increased with maturation of juvenile fronds of Enteromorpha sp. in culture. Thus, cultivation of seaweeds for flavor foods is of interest. The release of long chain aldehydes from the thallus into the medium was confirmed by a quantitative high performance liquid chromatography of volatile compounds, using a closed loop stripping technique, during the culture of the green alga, Ulva pertusa. This finding suggests physiological roles of long chain aldehydes and LCAE activity in marine ecosystems.     

Effect of angiotensin coverting enzyme inhibitor on regression in cardiac hypertrophy

Cardiac hypertrophy in rats was produced by aortic banding for 6 weeks and regression of hypertrophy in these experimental animals was induced by administration of angiotensin converting enzyme inhibitor, enalapril (10 mg/kg/ day) for 6 weeks. The left ventricular muscle mass and systolic pressure were decrease upon treating the hypertrophied rats with enalapril. This drug also decreased the number of 1-adrenoceptors in hypertrophyied myocardium without any changes in -adrenoceptors. The regression of cardiac hypertrophy in spontaneously hypertensive rats by enalapril for 10 weeks was not associated with any alterations in 1-adrenoceptors in hypertrophied myocardium, but was decreased in -adrenoceptors. Effects of enalapril on extracellular matrix in the myocardium was also observed in regression of hypertrophy in which the type III collagen mRNA expression and collagen contents were reduced in comparison with those of hypertrophied myocardium. These results indicate that regression of cardiac hypertrophy is not alway associated with a decrease in the number of 1-adrenergic receptors and that the beneficial effects of enalapril in the hypertrophied heart in aortic banding animals may be of some specific nature.     

MSl3, a multicopy suppressor of mutants hyperactivated in the RAS-CAMP pathway, encodes a novel HSP70 protein of Saccharomyces cerevisiae

The MSI3 gene was isolated as a multicopy suppressor of the heat shock-sensitive phenotype of the iral mutation, which causes hyperactivation of the RAS-cAMP pathway. Overexpression of MSI3 also suppresses the heat shock-sensitive phenotype of the bcyl mutant. Determination of the DNA sequence of MSI3 revealed that MSI3 can encode a 77.4 kDa protein related to the HSP70 family. The amino acid sequence of Msi3p is about 30% identical to that of the Ssalp of Saccharomyces cerevisiae. This contrasts with the finding that members of the HSP70 family generally show at least 50% amino acid identity. The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of MSI3. Moreover, the steady-state levels of the MSI3 mRNA and protein were increased upon heat shock. These results indicate that the MSI3 gene encodes a novel HSP70-like heat shock protein. Disruption of the MSI3 gene was associated with a temperature sensitive growth phenotype but unexpectedly, thermotolerance was enhanced in the disruptant.     

Noninvasive assessment of cardiac contractility by using (dP/dt)/P of carotid artery pulses during exercise

In earlier studies we have shown that both the pressure (P) of the carotid artery pulse (CAP) and its first derivative (CAP dP/dt) could be recorded during moderate exercise. To establish that the CAP (dP/dt)/P is a noninvasive substitute for the left ventricular (LV) value, LV (dP/dt)/P, an index of cardiac contractility, we studied CAP (dP/dt)/P under various states of activity in the autonomic nervous system in 12 healthy male subjects. Increased sympathetic nerve activities yielded by passive tilting, emotional load, or cold stress increased CAP (dP/dt)/P significantly (P< 0.05). Increased parasympathetic nerve activity by ocular compression, however, did not significantly affect the value. Moderate exercise at a heart rate of approximately 150 beats·min^–1 increased it significantly from 16.7 to 25.2·s^–1 in a supine position (P<0.001) and from 16.6 to 24.8·s^–1 in an upright position (P<0.001). It increased monotonically as heart rate increased, but the slope was steeper when the heart rate was greater than approximately 100 beats·min^–1 than it was when the rate was less than 100 beats·min^–1. In conclusion, the present study indicated that CAP (dP/dt)/P can be used as a noninvasive index of cardiac contractility even in moderate exercise.