Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.     

Continuous subcutaneous injection reduces polymorphonuclear leukocyte activation by granulocyte colony-stimulating factor

The use of granulocyte colony stimulating factor (G-CSF) for recovery from neutropenia has been established; however, acute lung injury due to G-CSF-induced polymorphonuclear leukocyte (PMN) activation is a serious complication. This study was designed to compare the activation of PMN with single bolus administration and continuous administration of G-CSF. Healthy volunteers (age 33.8 +/- 1.4 yr; n = 6) received a single bolus injection of 50 microm/m2 of G-CSF (SI; n = 6) or continuous subcutaneous injection of 50 microm/m2 of G-CSF for 24 h (CI; n = 6) and were followed for 48 h. Circulating leukocyte counts, markers of activation on PMN, and circulating levels of G-CSF, IL-6, and PMN elastase were measured. SI rapidly increased serum G-CSF levels, which peaked at 4 h, whereas CI gradually increased G-CSF levels, which remained at a steady level from 8 to 24 h. SI caused a rapid decrease in PMN counts at 0.5 h followed by sustained increase to peak at 12 h. CI gradually increased PMN counts, which peaked at 24 h, but the peak values were not significantly different between the groups. SI-induced activation of PMN, which was characterized by increased expression of CD11b, decreased expression of L-selectin, and increased F-actin content, led to increases in serum IL-6 and PMN elastase level. Such changes were all attenuated with CI (P < 0.05). We conclude that continuous subcutaneous injection of G-CSF resulted in a marrow response similar to that to a single injection but yielded reduced PMN activation.     

Exposure to ambient particles accelerates monocyte release from bone marrow in atherosclerotic rabbits

Exposure to air pollution [particulate matter, particles <10 microm (PM(10))] causes a systemic inflammatory response that includes stimulation of the bone marrow (BM) and progression of atherosclerosis. Monocytes are known to play a key role in atherogenesis by migration into subendothelial lesions where they appear as foam cells. The present study was designed to quantify the BM monocyte response in Watanabe heritable hyperlipidemic (WHHL) rabbits after PM(10) exposure. WHHL rabbits were given twice weekly intrapharyngeal instillations of 5 mg of PM(10) for 4 wk to a total of 40 mg and compared with control WHHL or New Zealand White (NZW) rabbits. The thymidine analog 5'-bromo-2'-deoxyuridine was used to label dividing cells in the BM and a monoclonal antibody to identify monocytes in peripheral blood. The transit time of monocytes through the BM was faster in WHHL than in NZW rabbits (30.4 +/- 1.9 h vs. 35.2 +/- 0.9 h, WHHL vs. NZW; P < 0.05). PM(10) instillation exposure increased circulating band cell counts, caused rapid release of monocytes from the BM, and further shortened their transit time through the BM to 23.2 +/- 1.6 h (P < 0.05). The percentage of alveolar macrophages containing particles in the lung correlated with the BM transit time of monocytes (r(2) = 0.45, P <0.05). We conclude that atherosclerosis increases the release of monocytes from the BM, and PM(10) exposure accelerates this process in relation to the amount of particles phagocytosed by alveolar macrophages.     

Gender difference regarding selenium penetration into the mouse brain

A sex difference in the penetration of selenium into the brain was observed using lipopolysaccharide (LPS)-injected mice. The selenium concentration increased in the brains of sodium selenite-injected LPS-treated female mice, but not males. The selenium concentration peaked when selenite was injected 3 h after the injection of LPS into female mice. In addition, selenium in the brain increased when a dosage of 30 μmol/kg and more of selenite was injected into LPS-treated female mice. Also, the selenium concentration in the brain increased and peaked 2–3 h after selenite injection; 24 h later, the level was similar to the Se-only group. The penetration of selenium into the brain was inhibited by pretreatment with aminoguanidine, an inhibitor of nitric oxide synthetase. From the present results, selenium more easily penetrated into the brains of female mice compared to males after LPS treatment, and nitric oxide may have affected the penetration. However, the sex difference mechanism for selenium penetration needs further investigation.     

Cloning and expression of the Momordica charantia trypsin inhibitor II gene in silkworm by using a baculovirus vector

MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei x Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI-II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4 x 10(-10)M for MCTI-II A and 5.2 x 10(-10) M for MCTI-II B.     

Recovery of Bacillus thuringiensis from Marine Sediments of Japan

Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions. Received: 29 November 1999 / Accepted: 10 January 2000     

Production of ubiquinone-10 using bacteria

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.     

Enhancement of Sodium Current in NG108-15 Cells during Neural Differentiation is Mainly due to an Increase in NaV1.7 Expression

It is well known that morphological and functional changes during neural differentiation sometimes accompany the expression of various voltage-gated ion channels. In this work, we investigated whether the enhancement of sodium current in differentiated neuroblastoma × glioma NG108-15 cells treated with dibutyryl cAMP is related to the expression of voltage-gated sodium channels. The results were as follows. (1) Sodium current density on peak voltage in differentiated cells was significantly enhanced compared with that in undifferentiated cells, as detected by the whole-cell patch clamp method. The steady-state inactivation curve in differentiated cells was similar to that for undifferentiated cells, but a hyperpolarized shift in the activation curve for differentiated cells was observed. The sodium currents of differentiated and undifferentiated cells were completely inhibited by 10⁻⁷ M tetrodotoxin (TTX). (2) The only Na_V mRNA with an increased expression level during neuronal differentiation was that for Na_V1.7, as observed by real-time PCR analysis. (3) The increase in the level of Na_V1.7 α subunit expression during neuronal differentiation was also observed by immunocytochemistry; in particular, the localization of Na_V1.7 α subunits on the soma, varicosities and growth cone was significant. These results suggest that the enhancement of TTX-sensitive sodium current density in differentiated NG108-15 cells is mainly due to the increase in the expression of the TTX-sensitive voltage-gated Na⁺ channel, Na_V1.7.