Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice

To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice.     

Low frequency fatigue in human quadriceps is fatigue dependent and not task dependent.

It is well accepted that a low intensity/long duration isometric contraction induces more low frequency fatigue (LFF) compared to a high-intensity/short-duration contraction. However, previous reports examined the intensity/duration of the contraction but did not control the level of fatigue when concluding fatigue is task dependent. The purpose of this study was to determine whether a long duration/low intensity fatiguing contraction would induce greater LFF than a short duration/high-intensity contraction when the quadriceps muscle was fatigued to similar levels. Eighteen healthy male subjects performed quadriceps contractions sustained at 35% and 65% of maximal voluntary contraction (MVC) on separate days, until the tasks induced a similar amount of fatigue (force generating capacity=45% MVC). Double pulse torque to single pulse torque ratio (D/S ratio) was obtained before, immediately and 5min after fatigue along with the electromyographic (EMG) signal from vastus medialis (VM) and rectus femoris (RF). The D/S ratio significantly (p<0.05) increased by 8.7+/-8.5% (mean+/-SD) and 10.2+/-9.2% after 35% and 65% tasks, respectively, and remained elevated 5min into recovery; however, there was no significant difference in ratio between the two sessions immediately or 5min post-fatigue (p>0.05) even though the endurance time for the 35% fatigue task (124+/-39.68s) was significantly longer (p=0.05) than that of the 65% task (63+/-17.73s). EMG amplitude and median power frequency (MPF) analysis also did not reveal any significant differences between these two sessions after fatigue. These findings indicate that LFF fatigue is fatigue dependent as well as task intensity/duration dependent. These findings assist us in understanding task dependency and muscle fatigue.     

Characterization of Green Tissue-Specific Phytochrome Isolated Immunochemically from Pea Seedlings

Phytochrome was isolated and purified from light-grown pea (Pisumsativum) seedlings and compared with that from dark-grown seedlingsin terms of spectral and immunochemical properties. Approximately40% of phytochrome in the brushite eluate prepared from light-grownpea tissue bound with a monoclonal anti-pea phytochrome antibody(mAP3), but the remaining 60% did not. Both phytochrome fractionsshowed a typical photoreversible absorbance change after alternatered and far-red actinic irradiations, which was similar to thatof phytochrome from etiolated pea tissue. The peptide mappingof the mAP3-bound phytochrome from light-grown tissue was essentiallythe same as that of the mAP3-bound phytochrome from etiolatedtissue. However, the digestion pattern of the phytochrome thatwas prepared from light-grown tissue but which did not bindto mAP3 was obviously different from that of mAP3-bound phytochrome.Polyclonal anti-pea phytochrome antibodies and mAP5 and 10,however, bound to both the phytochromes. These results suggestthat light-grown tissue contains two phytochrome pools whichare distinct from each other with respect to the primary structureof the phytochrome polypeptide but which share a few commondeterminant sites. ¹ Permanent address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa, Tokyo 158, Japan (H.A.), and Department of Botany, Faculty of Science, Universityof Tokyo, Hongo, Tokyo 113, Japan (M. F.).     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg²⁺-ATPase activity and the initial P_i burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system.

Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.