LIM kinase 1 modulates opsonized zymosan-triggered activation of macrophage-like U937 cells. Possible involvement of phosphorylation of cofilin and reorganization of actin cytoskeleton.

We have previously reported that cofilin, an actin-binding protein, plays an important role in phagocyte functions, such as respiratory burst, phagocytosis, and chemotaxis. On the other hand, it was recently found that LIM motif-containing kinase (LIMK) phosphorylates cofilin. In this work, we investigated the roles of LIMK in activated phagocytes. The results of immunostaining showed that in dormant phagocytes the endogenous LIMK1 was diffusely distributed in the cytosol of macrophage-like U937 cells, and when activated by opsonized zymosan (OZ), it was translocated to plasma membranes. Green fluorescence protein (GFP)-conjugated LIMK was expressed in the phagocytes, and the GFP-positive cells were isolated by a fluorescence-activated cell sorter. The isolated wild-type LIMK-overexpressing cells produced superoxide at a rate that was 3.2-fold higher than that of only GFP-expressing control cells, whereas the respiratory burst of dominant negative LIMK1(D460A)-expressing cells decreased to 31% of that of the control cells. Phagocytic activity monitored by using Texas Red-labeled OZ was also decreased in the D460A-expressing cells. By immunoblotting using a specific anti-phosphorylated cofilin antibody, it was revealed that in the OZ-activated wild-type LIMK1-GFP-expressing cells, the phosphorylated cofilin increased by 2.3-fold, and that in the OZ-activated D460A-GFP-expressing cells, the phosphorylated cofilin decreased to 47% of that of only GFP-expressing cells (mock control). Furthermore, in the wild-type LIMK1-expressing cells, OZ-evoked increase in filamentous actin was markedly enhanced, whereas in the dominant negative LIMK1-expressing cells, the total level of F-actin was strongly suppressed. These results suggest that LIMK1 regulates the functions of phagocytes through phosphorylation of cofilin and enhances the formation of filamentous actin.     

Effects of some naturally occurring iron ion chelators on in vitro superoxide radical formation

The effects of some naturally occurring iron ion chelators and their derivatives on the electron transfer from ferrous ions to oxygen molecules were examined by measuring oxygen consumption rates. Of the compounds examined, quinolinic acid, fusaric acid, and 2-pyridinecarboxylic acid repressed the oxygen consumption, whereas chlorogenic acid, caffeic acid, gallic acid, catechol l-β-(3,4-dihydroxyphenyl) alanine, and xanthurenic acid accelerated it. Theoretical calculations showed that the energies of the highest occupied molecular orbitals (HOMOs) of [Fe(II)(ligand)₃]⁻ complexes were relatively high when the ligands were caffeic acid and its derivatives such as catechol, gallic acid, and l-β-(3,4-dihydroxyphenyl) alanine. On the other hand, the energies of the HOMOs of [Fe(II)(ligand)₃]⁻ complexes were relatively low when the ligands were quinolinic acid and its derivatives such as 2-pyridinecarboxylic acid and fusaric acid. The energies of the HOMOs appear to be closely related with acceleration or repression of the oxygen consumption; that is to say, when the energy of the HOMO is high, the oxygen consumption is accelerated, and vice versa.     

Involvement of stress kinase mitogen-activated protein kinase kinase 7 in regulation of mammalian circadian clock

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.     

Spermatogenesis in the testes of diapause and non-diapause pupae of the sweet potato hornworm, Agrius convolvuli (L.) (Lepidoptera: Sphingidae)

Dichotomous spermatogenesis was examined in relation to diapause in the sweet potato hornworm, Agrius convolvuli. In non-diapause individuals, eupyrene metaphase began during the fifth larval instar and eupyrene spermatids appeared in wandering larvae. Bundles of mature sperm were found after pupation. Apyrene spermatocytes also appeared during the fifth larval instar, but meiotic divisions occurred irregularly and their nuclei were discarded from the cells during spermiogenesis. Morphometric analyses of flagellar axonemes showed a variable sperm number in apyrene bundles. The variation ranging from 125 to 256 sperm per bundle indicated abnormal divisions or the elimination of apyrene spermatocytes. In diapause-induced hornworms, spermatogenesis progressed similarly during the larval stages. The cessation of spermatogenesis during diapause is characterized by 1) secondary spermatocytes and sperm bundles degenerating gradually as the diapause period lengthens, and 2) spermatogonia or primary spermatocytes appearing throughout diapause. A TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay revealed that DNA fragmentation occurred in the nuclei of secondary spermatocytes and early spermatids. Aggregates of heterochromatin along the nuclear membrane indicated the onset of apoptosis, and condensed chromatin was confirmed by electron microscopy to be the apoptotic body. These results show that the degenerative changes in spermatogenic cells during pupal diapause were controlled by apoptosis.     

Cutaneous photobiology. The melanocyte vs. the sun: who will win the final round?

Solar ultraviolet radiation (UV) is a major environmental factor that dramatically alters the homeostasis of the skin as an organ by affecting the survival, proliferation and differentiation of various cutaneous cell types. The effects of UV on the skin include direct damage to DNA, apoptosis, growth arrest, and stimulation of melanogenesis. Long-term effects of UV include photoaging and photocarcinogenesis. Epidermal melanocytes synthesize two main types of melanin: eumelanin and pheomelanin. Melanin, particularly eumelanin, represents the major photoprotective mechanism in the skin. Melanin limits the extent of UV penetration through the epidermal layers, and scavenges reactive oxygen radicals that may lead to oxidative DNA damage. The extent of UV-induced DNA damage and the incidence of skin cancer are inversely correlated with total melanin content of the skin. Given the importance of the melanocyte in guarding against the adverse effects of UV and the fact that the melanocyte has a low self-renewal capacity, it is critical to maintain its survival and genomic integrity in order to prevent malignant transformation to melanoma, the most fatal form of skin cancer. Melanocyte transformation to melanoma involves the activation of certain oncogenes and the inactivation of specific tumor suppressor genes. This review summarizes the current state of knowledge about the role of melanin and the melanocyte in photoprotection, the responses of melanocytes to UV, the signaling pathways that mediate the biological effects of UV on melanocytes, and the most common genetic alterations that lead to melanoma.     

The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H₂O₂. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.     

Synthesis of creatol, a hydroxyl radical adduct of creatinine and its increase by puromycin aminonucleoside in isolated rat hepatocytes

Creatol is a hydroxyl radical adduct of creatinine and the precursor of methylguanidine (MG), a uremic toxin. We investigate the synthesis of creatol and MG from creatinine and the effect of substances that affect the hydroxyl radical in isolated rat hepatocytes. In the presence of increasing concentrations of creatinine, rising level of creatol were found after 2 h incubation in Krebs-Henseleit bicarbonate buffer. However, further increase of creatol was not observed after 4 and 6 h incubations. On the other hand, MG after 2 h incubation achieved a level of about 50% that of creatol and increased depending on both the creatinine concentration and the incubation period. DMSO, a hydroxyl radical scavenger decreased the generation of creatol and MG by about 50% at 2.5 mM and the inhibition depended on DMSO concentration. Puromycin aminonucleoside (PAN) increased both by about 170%. These findings demonstrated that hepatocytes synthesize creatol prior to MG and are inhibited by a hydroxyl radical scavenger. They also show that PAN increased hydroxyl radical generation in tissue cells.     

Plant regeneration via somatic embryogenesis from protoplasts of Uganda cherry orange (Citropsis schweinfurthii)

Protoplasts isolated from embryogenic callus of Citropsis schweinfurthii (Engl.) Swing. & M. Kell were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l^–1 BA, 0, 300, 600 or 900 mg l^–1 malt extract and 0.6 M sorbitol. The highest plating efficiency was obtained on MT basal medium containing 5% sucrose supplemented with 0.01 mg l^–1 BA and 600 mg l^–1 malt extract. MT basal medium containing 5% sucrose and supplemented with 0.01 mg l^–1 kinetin was found to be a medium suitable for the development of globular somatic embryos derived from protoplasts into heart-shaped somatic embryos with cotyledon-like structures. The highest percentage of shoot formation was obtained using 0.1 mg l^–1 GA₃. A complete protoplast to-plant system was developed for C. schweinfurthii, which could facilitate the transfer of nuclear and cytoplasmic genes from this species into cultivated Citrus through protoplast fusion.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellin A₃ - ME malt extract     

p250GAP,a novel brain-enriched GTPase-activating protein for Rho family GTPases,is involved in the N-methyl-d-aspartate receptor signaling

N-methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRepsilon2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRepsilon2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.