Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

Effects of inhibitors and NaCl on the oxidation of reduced inorganic sulfur compounds by a marine acidophilic, sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH

The effect of NaCl and the pathways of the oxidation of reduced inorganic sulfur compounds were studied using resting cells and cell-free extracts of Acidithiobacillus thiooxidans strain SH. This isolate specifically requires NaCl for growth. The oxidation of sulfur and sulfite by resting cells was strongly inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. Carbonylcyanide m-chlorophenyl-hydrazone and monensin were also relatively strong inhibitors. Thiosulfate-oxidizing activity was not inhibited by these uncouplers. Valinomycin did not inhibit the oxidation of sulfur compounds. NaCl stimulated the sulfur- and sulfite-oxidizing activities in resting cells but not in cell-free extracts. The tetrathionate-oxidizing activity in resting cells was slightly stimulated by NaCl, whereas it did not influence the thiosulfate-oxidizing activity. Sulfide oxidation was biphasic, suggesting the formation of intermediate sulfur. The initial phase of sulfide oxidation was not affected by NaCl, whereas the subsequent oxidation of sulfur in the second phase was Na⁺-dependent. A model is proposed for the role of NaCl in the metabolism of reduced sulfur compounds in A. thiooxidans strain SH.     

GINS is a DNA polymerase epsilon accessory factor during chromosomal DNA replication in budding yeast

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase epsilon (Pol epsilon) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol epsilon is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol epsilon in vitro. These results strongly suggest that GINS is a Pol epsilon accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork.     

Involvement of phosphorylation of beta-subunit in cAMP-dependent activation of L-type Ca2+ channel in aortic smooth muscle-derived A7r5 cells

We investigated the effect of intracellular cAMP on the gating kinetics of L-type Ca2+ channel in an A7r5 smooth muscle-derived cell line using the whole-cell patch-clamp technique. Application of dibutyryl cyclic AMP (db-cAMP) to the cell increased the magnitude of Ca2+ currents through L-type Ca2+ channels (I(Ca)), and shifted the current-voltage relationship (I-V curve) for I(Ca) to the left. The magnitudes of maximum I(Ca) were 14.1 +/- 0.7 before and 16.0 +/- 1.1 pA/pF after application of 1 mM db-cAMP (P < 0.05). The values of the half-activation potential (V(1/2)) of I(Ca), estimated from activation curves, were -7.0 +/- 0.8 mV before and -10.8 +/- 1.0 mV after application of db-cAMP (P < 0.05). In cells pretreated with 10 microM Rp-cAMPS (a specific inhibitor of PKA), db-cAMP affected neither the I-V curve nor the activation curve for I(Ca). In cells pretreated with the antisense oligonucleotide for the beta-subunit of L-type Ca2+ channel, db-cAMP failed to enhance I(Ca) or alter the activation curve. On the other hand, in the cells pretreated with the nonsense oligonucleotide, application of db-cAMP caused an increase in magnitude of I(Ca) and shifted the activation curve to the left. Western blot analysis revealed that the pretreatment of cells with antisense oligonucleotide but nonsense oligonucleotide reduced the expression of the beta-subunit of the L-type Ca2+ channel. We conclude that the cAMP-dependent phosphorylation of the beta-subunit potentiates the voltage dependency of the activation kinetics of the L-type Ca2+ channel in A7r5 cells.     

Clinico-pathological studies of LEC rats with hereditary hepatitis and hepatoma in the acute phase of hepatitis

The LEC rat, which suffers from hereditary hepatitis, was examined for elucidation of its clinicopathological characteristics during development of the acute phase of hepatitis by quantitative analyses of histological observations of the liver in combination with laboratory data on various serum enzymes. The progression of acute hepatitis in the LEC rat was observed to begin insidiously early in life, i.e., a few enlarged hepatocytes and Councilman bodies appeared at around 8 weeks of age without clinical signs. Furthermore, it was revealed that the acute phase of hepatitis started with a remarkable increase of Councilman bodies, large nuclei and hepatocytes in mitosis in the liver 3 to 4 weeks before the onset of fulminant hepatitis, which is characterized by the elevation of serum enzyme activities such as GOT, GPT and gamma-GTP, and the onset of jaundice. From those observations, three stages were proposed for the progression of acute hepatitis in the LEC rat.     

Effects of broken male intromittent organs on the sperm storage capacity of female earwigs, <Emphasis Type="Italic">Euborellia plebeja</Emphasis>

In earwigs of the family Anisolabididae, male intromittent organs (virgae) sometimes break off inside female sperm-storage organs (spermathecae) during mating. I examined the effects of this genital breakage on the sperm storage capacity of females using Euborellia plebeja as a representative species. When genital breakage was artificially induced in virgin females, subsequent males successfully inseminated these females. However the sperm-storage capacity of these females was limited by the presence of broken virgae in their spermathecae. In another experiment, genital breakage was experimentally induced in the spermathecae of inseminated females, and their reproductive performance was then monitored for 60 days. In all of four cases where the entire piece of the broken virga remained inside the spermatheca, females deposited fertile eggs (more than 60% hatchability). The average number of clutches, that of eggs laid, and that of hatchlings were similar to those of controls. On the other hand, females laid no eggs in the other two cases where the broken virgae protruded from the spermathecal opening. I discuss the relevance of the results to the mating system and possible removal of rival sperm, which has been reported for E. plebeja. Electronic Publication     

Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

Background

Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster.

Methodology/Principal Findings

To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids.

Conclusions/Significance

From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.     

A Vertical Gradient of the Chloroplast Abundance among Leaves of Chenopodium album

The abundances of chloroplasts in leaves on the main stems ofChenopodium album at different height levels were investigatedin relation to the photosynthetic capacity and light environmentof the leaves. (1) The number of chloroplasts per mesophyllcell decreased with descending position of leaves, except foryoung developing leaves at the top of plants that had smallerchloroplast numbers per cell than matured leaves beneath them.Contents of chlorophyll and ribulose-1,5-bisphosphate carboxylase/oxygenaseper leaf area that were highest in the topmost young leavesand decreased with decreasing height level indicate that thereis a vertical gradient of chloroplast abundance per leaf areadecreasing from the top of the leaf canopy with depth. (2) Light-saturatingrate of photosynthetic oxygen evolution per leaf area of maturedleaves decreased more steeply with decreasing leaf positionthan the chloroplast number per cell. Gradients of chlorophylland the enzyme protein contents were also steeper than thatof the chloroplast number. Loss of photosynthesis in lower leavesis, therefore, ascribed partly to loss of whole chloroplastsand partly to reduced photosynthetic capacities of the remainingchloroplasts. (3) The chloroplast number per cell in newly expandedsecond leaves was comparable to those in leaves that have developedat later stages of the plant growth but decreased graduallyduring leaf senescence both in the dark and light. The formationof the vertical gradient of chloroplast abundance is, therefore,ascribed to loss of whole chloroplasts during senescence ofleaves. (4) Irradiance a leaf receives decreased sharply fromthe top of the canopy with depth. The physiological or ecophysiologicalsignificance of the vertical distribution of chloroplasts amongleaves was discussed taking light environments of leaves intoconsideration. (Received July 31, 1995; Accepted October 20, 1995)     

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.     

IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130(F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.