Possible participation of calmodulin in stimulation of leucine transport by concanavalin A in human lymphocytes

Stimulation of leucine uptake by addition of concanavalin A, mediated by increase of intracellular free Ca2+ concentration [( Ca2+]), in lymphocytes (Mitsumoto, Y., Sato, K. and Mohri, T. (1988) Biochim. Biophys. Acta 968, 353-358) was abolished by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine, which inhibited membrane hyperpolarization induced by the mitogen. Quinine (0.5-1 mM) completely inhibited the concanavalin A-induced hyperpolarization and extensively inhibited the induced stimulation of leucine uptake. Based on these results, we suggest that the stimulation of leucine uptake by concanavalin A is largely due to activation of the Ca2+-dependent K+ channel which reinforces negative potential of the plasma membrane and is regulated by calmodulin.     

Acute and long-term effects of insulin-like growth factor I on glucose transporters in muscle cells. Translocation and biosynthesis.

Insulin-like growth factor I (IGF-I) rapidly (less than 10 min) stimulated glucose uptake into myotubes of the L6 muscle cell line, at concentrations that act specifically on IGF-I receptors. Uptake remained stimulated at a steady level for 1-2 h, after which a second stimulation occurred. The first phase was insensitive to inhibition of protein synthesis. Subcellular fractionation demonstrated that it was accompanied by translocation of glucose transporters (both GLUT1 and GLUT4) to the plasma membrane from intracellular membranes. Translocation sufficed to explain the first phase increase in glucose transport, and there was no change in the total cellular content of GLUT1 or GLUT4 glucose transporters. The second phase of stimulation was inhibitable by cycloheximide, and involved a net increase in either GLUT1 or GLUT4 transporter content, which was reflected in an increase in transporter number in plasma membranes. These results define a cellular mechanism of metabolic action of IGF-I in muscle cells; furthermore, they suggest that IGF-I has acute metabolic effects that mimic those of insulin, bypassing action on the insulin receptor.     

Fibroblast colonies in monolayer cultures of human bone marrow

In a liquid culture of human bone marrow, the development of fibroblast colonies takes place on days 6 to 9. Twenty percent fetal calf serum is used as the stimulus for fibroblast colony growth. Human bone marrow cells are plated as 2 × 10₅ cells in the culture. Normal human bone marrow yields 47 ± 4 fibroblasts colonies per 2 × 10₅ cells plated. Bone marrow fibroblast cultures using agar or methylcellulose restrict colony formation. Marked colony suppression was observed in acute leukemia, and a discrete colony number was observed in hypoplastic anemia. This fibroblast culture method should be applied to a larger number of patients to determine whether it has a pathognomonic value and clinical significance.     

Efficient synthesis and structure-activity relationship of honokiol, a neurotrophic biphenyl-type neolignan

Honokiol, a biphenyl-type neolignan, which shows the remarkable neurotrophic effect in primary cultured rat cortical neurons, has been effectively synthesized in 21% yield over 14 steps starting from 5-bromosalicylic acid and p-hydroxybenzoic acid by utilizing Pd-catalyzed Suzuki-Miyaura coupling reaction as a key step. Additionally, the structure-activity relationship between neurite outgrowth-promoting activity and its O-methylated and/or its hydrogenated analogues was examined in the primary cultures of fetal rat cortical neurons, suggesting that 5-allyl and 4'-hydroxyl groups are essential for affecting the neurotrophic activity of honokiol.     

Identification and characterization of TMEFF2, a novel survival factor for hippocampal and mesencephalic neurons

We have identified a novel mammalian gene, TMEFF2, that encodes a putative transmembrane protein containing two follistatin-like domains and one epidermal growth factor (EGF)-like domain. The TMEFF2 gene is predominantly expressed in the brain. In situ hybridization analysis revealed that TMEFF2 is widely expressed in the brain, including hippocampal cornu ammonis, dentate gyrus, and substantia nigra pars compacta. We evaluated the survival effect of TMEFF2 using primary cultured neurons from several regions of fetal rat brain following treatment with a recombinant TMEFF2 protein fragment consisting of the putative extracellular domain. TMEFF2 increased survival of neurons from the hippocampus and midbrain, but not from the cerebral cortex, indicating that the survival effects of TMEFF2 are specific to certain cell types. Recombinant TMEFF2 also promoted survival of mesencephalic dopaminergic neurons. Together, these findings suggest that TMEFF2 may be a novel survival factor for hippocampal and mesencephalic, but not for cortical, neurons.     

Evidence of cross-fertilization in a gonochoric population of the tadpole shrimp Triops numidicus (Crustacea: Branchiopoda: Notostraca)

Cross-fertilization was evident in a gonochoric population of the notostracan Triops numidicus, in which a thick, hard eggshell had been suspected of preventing the sperm from reaching the egg. Most of the eggs from copulated females hatched into larvae, but the eggs from virgin females did not develop. In the larvae, paternal DNA fragments were detected by AFLP. In histological sections, several spermatozoa were found in the shell of newly oviposited eggs in the brood pouches of copulated females, suggesting that the shell was still soft enough to be penetrated by spermatozoa. These results showed that copulation and subsequent fertilization achieved by penetration of sperm through the newly deposited eggshell were indispensable for the normal development of the eggs.     

Oxidized forms of peroxiredoxins and DJ-1 on two-dimensional gels increased in response to sublethal levels of paraquat

We previously found hydroperoxide-responsive proteins (HPRPs), which are comprised of peroxiredoxin I(Prx I), Prx II, Prx III, Prx VI, HSP27, G3PDH and two unidentified proteins (HPRP-2' and HPRP-5'), in human umbilical vein endothelial cells. It was demonstrated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) that most HPRPs are converted into variants with lower pI upon exposure to hydroperoxides. In this study, we examined the HPRP response on 2D gels upon exposure of human endothelial cells (ECV304) to paraquat (PQ²⁺), which generates reactive oxygen species (ROS) within cells. PQ²⁺ exerted cytotoxic effects in a dose- (10μM-10mM) and time- (24-168h) dependent manner. Two-dimensional PAGE analysis revealed that HPRP-2', and oxidized forms of Prx I, Prx II and Prx III were clearly increased upon exposure of cells to sublethal levels of PQ²⁺. Microsequence analysis revealed that both HPRP-2 and -2' were identical with human DJ-1. Moreover immunoblot analysis confirmed the increase of oxidized forms of Prx II, Prx III and DJ-1 in response to sublethal levels of PQ²⁺. PQ²⁺ treatment failed to increase fluorescence intensity derived from DCF, which is believed to be an indicator for intracellular levels of hydroperoxide. Although pentachlorophenol (PCP), an uncoupler of the mitochondrial respiratory chain, clearly elevated the fluorescence, PCP had no effect on HPRP response. These observations indicated that DCF-derived fluorescence is not correlated with HPRP response. We consider that the response of Prxs and DJ-1 on 2D gels could reflect endogenous production of ROS in PQ²⁺-treated cells, and might be a sensitive indicator of oxidative stress status.     

DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin.

We previously found that DJ-1 protein of pI 5.8 (DJ-1/5.8) increased on 2D gels as DJ-1 of pI 6.2 (DJ-1/6.2) decreased, upon exposure of human cells to sublethal levels of oxidative stress, such as H2O2 and paraquat. Here, we show that the DJ-1/5.8 increases concomitantly with endogenous production of reactive oxygen species (ROS) under endotoxin-induced inflammatory conditions. Lipopolysaccharide (LPS) significantly increased the expression of DJ-1/5.8 in murine peritoneal macrophages (Mphi) and a murine macrophage cell line (J774). Diphenylene iodonium, a flavoenzyme inhibitor, blocked the effect of LPS on DJ-1/5.8 expression. Aminoguanidine (AG), a selective inhibitor of type II nitric oxide synthase, had no effect on the DJ-1/5.8 expression, but suppressed accumulation of nitrite in the culture medium after LPS treatment. We also examined the expression of DJ-1/5.8 in lung, since acute lung injury is seen in endotoxin shock. When female mice (6-weeks old) were intraperitoneally given LPS (10 mg/kg), myeloperoxidase (MPO) activity in lung, a marker of neutrophil infiltration, was transiently raised by 3.5 fold. The expression of DJ-1/5.8 in lung was enhanced and then reverted to the control level, in parallel with the MPO activity. These results, taken together, suggest that the DJ-1/5.8 might increase in response to endogenously produced ROS, probably due to activation of NADPH oxidase, and imply that DJ-1 may be useful as an endogenous indicator of oxidative stress status in vivo.     

Leucine transport in relation to the activities of Na+-H+ antiporter and Na+/K+ pump stimulated by serum and a tumor promoter

Fetal calf serum and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the rate of leucine uptake by Chang liver cells in Na+-containing medium. Addition of monensin to the incubation medium also increased the leucine uptake. All these agents were capable of raising the cytoplasmic pH, which was blocked by a prior addition of amiloride or removing Na+ from assay medium, suggesting activation of Na+-H+ exchange across the cell membrane by fetal calf serum and TPA. The stimulation of leucine uptake by monensin and fetal calf serum was blocked completely or incompletely by addition of ouabain or amiloride. The basal and fetal-calf-serum- or TPA-stimulated leucine uptake was extensively depressed by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid in the incubation medium. Based on these results it is proposed that the transport of leucine by the system L is stimulated by fetal calf serum and TPA with a high concentration of Na+ outside the cells as a result of alkalinization of the cytoplasm and coordinated activation of (Na+ + K+)-ATPase by these stimulators to maintain the transmembrane Na+ gradient and also hyperpolarize the cell membrane.