Studies on the ontogenesis and the regulatory mechanisms of placental lactogen and insulin binding sites (receptor) in rat liver

The ontogenesis of specific binding of 125I-hPL and 125I-insulin was determined in rat liver cell membranes (10(5) X g pellets), and the regulatory mechanisms of these binding sites were also examined. There were striking differences in the mode of ontogenesis between binding sites of hPL and insulin in rats. HPL binding sites were very few in liver cell membranes from fetal and immature rats. They began to increase after puberty, and markedly increased in late pregnancy. On the other hand, insulin binding sites, which decreased in late pregnancy, were dominant in fetal liver and placenta. Consequently, the lipolytic and glycogenolytic activities of hPL in maternal liver were accentuated, whereas the effects of insulin on maternal liver were suppressed. In contrast, in fetal liver and placenta only the anabolic effects of insulin seemed conspicuous. According to the results of experiments on in vivo administration of estradiol-17 beta, progesterone, hydrocortisone or hPL to intact or hypox-rats, and the measurement of serum rat chorionic mammotropin (rCM), rPRL, estradiol-17 beta, and insulin during pregnancy in rats, the increase in hepatic hPL binding sites observed in late pregnancy might be, at least in part, due to rCM secreted from placenta, and the decrease in insulin binding sites due to the increase in serum insulin itself in rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ciliary Neurotrophic Factor (CNTF) Genotypes and CNTF Contents in Human Sciatic Nerves as Measured by a Sensitive Enzyme-Linked Immunoassay

Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme-linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.     

Intracellular Ca2+ increase induces post-fertilization events via MAP kinase dephosphorylation in eggs of the hydrozoan jellyfish Cladonema pacificum

Naturally spawned eggs of the hydrozoan jellyfish Cladonema pacificum are arrested at G1-like pronuclear stage until fertilization. Fertilized eggs of Cladonema undergo a series of post-fertilization events, including loss of sperm-attracting ability, expression of adhesive materials on the egg surface, and initiation of cell cycle leading to DNA synthesis and cleavage. Here, we investigate whether these events are regulated by changes in intracellular Ca2+ concentration and mitogen-activated protein kinase (MAP kinase) activity in Cladonema eggs. We found that MAP kinase is maintained in the phosphorylated form in unfertilized eggs. Initiation of sperm-induced Ca2+ increase, which is the first sign of fertilization, was immediately followed by MAP kinase dephosphorylation within a few minutes of fertilization. The fertilized eggs typically stopped sperm attraction by an additional 5 min and became sticky around this time. They further underwent cytokinesis yielding 2-cell embryos at approximately 1 h post-fertilization, which was preceded by DNA synthesis evidenced by BrdU incorporation into the nuclei. Injection of inositol 1,4,5-trisphosphate (IP3) into unfertilized eggs, which produced a Ca2+ increase similar to that seen at fertilization, triggered MAP kinase dephosphorylation and the above post-fertilization events without insemination. Conversely, injection of BAPTA/Ca2+ into fertilized eggs at approximately 10 s after the initiation of Ca2+ increase immediately lowered the elevating Ca2+ level and inhibited the subsequent post-fertilization events. Treatment with U0126, an inhibitor of MAP kinase kinase (MEK), triggered the post-fertilization events in unfertilized eggs, where MAP kinase dephosphorylation but not Ca2+ increase was generated. Conversely, preinjection of the glutathione S-transferase (GST) fusion protein of MAP kinase kinase kinase (Mos), which maintained the phosphorylated state of MAP kinase, blocked the post-fertilization events in fertilized eggs without preventing a Ca2+ increase. These results strongly suggest that all of the three post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle, lie downstream of MAP kinase dephosphorylation that is triggered by a Ca2+ increase.     

Mutations affecting retina development in Medaka

In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation.     

Protracted Administration of L-Asparaginase in Maintenance Phase Is the Risk Factor for Hyperglycemia in Older Patients with Pediatric Acute Lymphoblastic Leukemia

Although L-asparaginase related hyperglycemia is well known adverse event, it is not studied whether the profile of this adverse event is affected by intensification of L-asparaginase administration. Here, we analyzed the profile of L-asparaginase related hyperglycemia in a 1,176 patients with pediatric acute lymphoblastic leukemia treated according to the Japan Association of Childhood Leukemia Study ALL-02 protocol using protracted L-asparaginase administration in maintenance phase. We determined that a total of 75 L-asparaginase related hyperglycemia events occurred in 69 patients. Although 17 events (17/1176, 1.4%) developed in induction phase, which was lower incidence than those (10–15%) in previous reports, 45 events developed during the maintenance phase with protracted L-asparaginase administration. Multivariate analysis showed that older age at onset (≥10 years) was a sole independent risk factor for L-asparaginase-related hyperglycemia (P<0.01), especially in maintenance phase. Contrary to the previous reports, obesity was not associated with L-asparaginase-related hyperglycemia. These findings suggest that protracted administration of L-asparaginase is the risk factor for hyperglycemia when treating adolescent and young adult acute lymphoblastic leukemia patients.     

Spatiotemporal analysis of Ca(2+) waves in relation to the sperm entry site and animal-vegetal axis during Ca(2+) oscillations in fertilized mouse eggs

Fertilized mouse eggs exhibit repetitive rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) necessary for egg activation. Precise spatiotemporal dynamics of each [Ca(2+)](i) rise were investigated by high-speed Ca(2+) imaging during early development of monospermic eggs. Every [Ca(2+)](i) rise involved a Ca(2+) wave. In the first Ca(2+) transient, [Ca(2+)](i) increased in two steps separated by a "shoulder" point, suggesting two distinct Ca(2+) release mechanisms. The first step was a Ca(2+) wave that propagated from the sperm-fusion site to its antipode in 4-5 s (velocity, approximately 20 microm/s in most eggs). The second step from the shoulder to the peak was a nearly uniform [Ca(2+)](i) rise of 12-15 s. A slight cytoplasmic movement followed the Ca(2+) wave in the same direction and recovered in 25-35 s. These characteristics changed as follows, as Ca(2+) oscillations progressed during the second meiosis up to their cessation at the stage of pronuclei formation ( approximately 3 h after fertilization). (1) The duration of Ca(2+) transients became shorter. (2) The shoulder point shifted to higher levels and the first step occupied most of the rising phase. (3) The rate of [Ca(2+)](i) rise became greater and wave speeds increased up to 80-100 microm/s or more. (4) The transient cytoplasmic movement always resulted from the Ca(2+) wave, although its displacement became smaller. (5) The Ca(2+) wave initiation site was freed from the sperm-fusion or -entry site and eventually localized in the cortex of the vegetal hemisphere. Since the shift of the wave initiation site to the vegetal cortex is observed in fertilized eggs of nemertean worms and ascidians, this might be an evolutionarily conserved feature.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.     

Spatiotemporal characteristics and mechanisms of intracellular Ca(2+) increases at fertilization in eggs of jellyfish (Phylum Cnidaria, Class Hydrozoa)

We have clarified, for the first time, the spatiotemporal patterns of intracellular Ca(2+) increases at fertilization and the Ca(2+)-mobilizing mechanisms in eggs of hydrozoan jellyfish, which belong to the evolutionarily old diploblastic phylum, Cnidaria. An initial Ca(2+) increase just after fertilization took the form of a Ca(2+) wave starting from one cortical region of the egg and propagating to its antipode in all of four hydrozoan species tested: Cytaeis uchidae, Cladonema pacificum, Clytia sp., and Gonionema vertens. The initiation site of the Ca(2+) wave was restricted to the animal pole, which is known to be the only area of sperm-egg fusion in hydrozoan eggs, and the wave propagating velocity was estimated to be 4.2-5.9 mum/s. After a Ca(2+) peak had been attained by the initial Ca(2+) wave, the elevated Ca(2+) gradually declined and returned nearly to the resting value at 7-10 min following fertilization. Injection of inositol 1,4,5-trisphosphate (IP(3)), an agonist of IP(3) receptors (IP(3)R), was highly effective in inducing a Ca(2+) increase in unfertilized eggs; IP(3) at a final intracellular concentration of 12-60 nM produced a fully propagating Ca(2+) wave equivalent to that observed at fertilization. In contrast, a higher concentration of cyclic ADP-ribose (cADPR), an agonist of ryanodine receptors (RyR), only generated a localized Ca(2+) increase that did not propagate in the egg. In addition, caffeine, another stimulator of RyR, was completely without effect. Sperm-induced Ca(2+) increases in Gonionema eggs were severely affected by preinjection of heparin, an inhibitor of Ca(2+) release from IP(3)R. These results strongly suggest that there is a well-developed IP(3)R-, but not RyR-mediated Ca(2+) release mechanism in hydrozoan eggs and that the former system primarily functions at fertilization. Our present data also demonstrate that the spatial characteristics and mechanisms of Ca(2+) increases at fertilization in hydrozoan eggs resemble those reported in higher triploblastic animals.     

Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.     

Molecular cloning and expression of the col2a1a and col2a1b genes in the medaka, Oryzias latipes

The Col2a1 gene is expressed in notochord, otic vesicle, cartilaginous tissue and the anlage of endochondral bone during development in higher vertebrates. Type II collagen, a homotrimeric product of the Col2a1 gene, functions as a key regulatory protein for cartilage development and endochondral ossification. In medaka and zebrafish, a single homolog of the col2a1 gene has been identified. However, it is necessary to note that many genes are duplicated in teleost fishes. To clarify function of col2a1 genes in teleost fishes and to further understand the process of cartilage development and endochondral ossification, we cloned and mapped the gene loci of two col2a1 orthologs in medaka. The proteins encoded by both medaka col2a1a and col2a1b genes were highly conserved (85.3% and 82.6%) relative to human COL2A1, but synteny was not observed. We also examined the expression patterns of col2a1a and col2a1b during embryonic development. Whole-mount insitu hybridization data suggests that expression patterns of both medaka co2a1a and col2a1b genes are similar to that of zebrafish co2a1 in the early embryonic stages. In medaka, the two col2a1 genes show a closely correlated pattern of spatial and temporal expression. In late embryonic stages, however, there were differences in both expression patterns in the pectoral fin. This study is the first report of two homologs of col2a1 in teleosts and also the first examination of col2a1a and col2a1b expression patterns in this group.