Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis

Cd induces oxidative stress and apoptosis in various cells by activating mitogen-activated protein kinases (MAPKs), but the precise signaling components of the MAPK cascade and their role in neuronal apoptosis are still unclear. Here, we report that Cd treatment of SH-SY5Y cells caused apoptosis through sequential phosphorylation of the apoptosis signal regulating kinase 1, MAPK kinase 4, c-Jun N-terminal kinase (JNK), and c-Jun as determined by overexpression of dominant negative (DN) constructs of these genes or using a specific JNK inhibitor SP600125. Both Cd-induced JNK and c-Jun phosphorylation and apoptosis were inhibited dramatically by N-acetyl-L-cysteine, a free radical scavenger. In addition, caspase inhibitors, zDEVD and zVAD, reduced apoptosis but not JNK and c-Jun phosphorylation induced by Cd, while overexpression of DN JNK1 inhibited caspase-3 activity. Taken together, our data suggested that the JNK/c-Jun signaling cascade plays a crucial role in Cd-induced neuronal cell apoptosis and provides a molecular linkage between oxidative stress and neuronal apoptosis.     

Reversible regulation of P2Y(2) nucleotide receptor expression in the duct-ligated rat submandibular gland

Ligation of the main excretory duct of the rat submandibular gland(SMG) produces a pronounced atrophy that is reversed upon ligatureremoval. Based on previous studies by our group and others suggestingthat P2Y₂ nucleotide receptors are upregulated in response to tissue damage, we hypothesized that P2Y₂ receptoractivity and mRNA levels would increase after duct ligation and return to control levels after ligature removal. Our results support thishypothesis. Intracellular Ca²⁺ mobilization in response tothe P2Y₂ receptor agonist UTP in SMG cells was increasedsignificantly after ligation periods of 1.5 to 7 days, whereas nosignificant response was observed in the contralateral, nonligatedgland. P2Y₂ receptor mRNA, as measured bysemiquantitative RT-PCR, increased about 15-fold after 3 days ofligation. These increases reverted to control levels by 14 days afterligature removal. In situ hybridization revealed that the changes inP2Y₂ receptor mRNA abundance occurred mostly in acinarcells, which also were more adversely affected by ligation, includingan increase in the appearance of apoptotic bodies. These findingssupport the idea that P2Y₂ receptor upregulation may be animportant component of the response to injury in SMG and that recoveryof normal physiological function may signal a decreased requirement forP2Y₂ receptors.

     

The roles of sterol regulatory element-binding proteins in the transactivation of the rat ATP citrate-lyase promoter

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from -114 to -60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the -102/-94 region, immediately upstream of the inverted Y-box, and the -70/-61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter.     

Effect of DNA topology on plasmid DNA repair in vivo

Escherichia coli nucleotide excision repair (NER) is responsible for removing bulky DNA adducts by dual incisions of the UvrABC endonuclease. Although the activity of the UvrAB complex which can induce DNA conformational change is employed in NER, the involvement of DNA topology and DNA topoisomerases remains unclear. We examined the effect of topoisomerase inhibitions on a NER in vivo system. The repair analysis of intracellular plasmid revealed that the DNA damage on positive supercoils generated by gyrase inhibition remained unrepaired, whereas the DNA damage was repaired in topoisomerase I mutants. These results suggest that DNA topology affects the NER process and the removal of positive supercoils by gyrase is vital for the efficiency of the E. coli NER system.     

The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.     

A systemic administration of NMDA induces immediate early gene pip92 in the hippocampus

Abstract : In the mammalian CNS, aspartate and glutamate are major excitatory amino acids, and their receptors are believed to mediate a wide range of physiological and pathological processes, including neurotransmission, plasticity, excitotoxicity, and various forms of neurodegeneration. The immediate early gene pip92 has been identified in serum-stimulated BALB/c 3T3 fibroblasts, activated T lymphocytes treated with cycloheximide, and fibroblast growth factor-stimulated hippocampal cells during neuronal differentiation. In this study we have demonstrated that pip92 is expressed in the mouse brain after a single intraperitoneal injection of NMDA. The distribution of pip92 mRNA levels in the NMDA-treated mouse brain was investigated using in situ RT-PCR. The region-specific activation of pip92 in the CNS was observed 3 h after NMDA injection, and high levels of pip92 mRNA were detected in the hippocampal dentate gyrus and piriform cortex regions. In addition, the activation of pip92 by NMDA was mediated by activation of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK) and p38 kinase, but not extracellular signal-regulated kinase (ERK) in the mouse hippocampus and immortalized rat hippocampal progenitor cells. This study suggests that pip92 is likely to play an important role in neuronal cell death induced by excitotoxic NMDA injury in the CNS.     

Infestation status of head louse and treatment with lindane shampoo in children of primary school and kindergarten in Chinju-shi, Kyongsangnam-do, Korea

The infestation status of head louse among children attending primary schools and kindergartens in Chinju-shi, Kyongsangnam-do, Korea, was investigated between June and July 1999. Out of 2,288 children examined, 3.9% of boys (48/1,242) and 23.5% of girls (246/1,046) were infested with nits or adult/nymphs of lice. The effectiveness of lindane shampoo (1% gamma benzene hexachloride solution) was evaluated after one or two time applications to all the children infested. The negative conversion rate of pediculosis was 93.5%. Effective control measures are needed to control and prevent such ectoparasite infestation amongst children.     

Botulinum toxin A for the treatment of facial hyperkinetic wrinkle lines in Koreans

There are a number of different causes for facial wrinkle lines, such as aging, gravity, and chronic pulling of mimetic muscles on the face. Among these, pulling by mimetic muscles on the skin not only involves facial expression but also has a great role in forming facial wrinkle lines as a result of repetitive action, such as dynamic or hyperkinetic wrinkle lines. Botulinum toxin A is currently being used for eliminating facial hyperkinetic wrinkles by causing paralysis of the underlying mimetic muscles. Because there are some histologic differences between Asians and Caucasians, such as thick dermis and more abundant collagen fiber, etc., the chronic pulling by mimetic muscles on the skin is expected to affect facial wrinkles differently. Therefore, the purpose of this study was to determine the efficacy of botulinum toxin A injection in eliminating facial hyperkinetic wrinkle lines among Korean patients. This study included 38 patients and 59 injection sessions from January of 1996 to April of 1997. We used Botox containing 100 U. Toxin was diluted with 4 ml of sterile normal saline and yielded 2.5 U for each 0.1 cc. A dose of 5 to 10 U was used in each muscle. Ages ranged from 26 to 56 years. There were 33 women and 5 men included in this study. Thirty-two of the patients were followed from 3 months up to 12 months after injections. The number of injection sessions that were performed on each patient was as follows: one session, 23 patients; two sessions, 10 patients; three sessions, 4 patients; four sessions, 1 patient. The number of injections per target site among these 38 patients was as follows: lateral canthal area, 33; glabellar area, 9; forehead, 9; nasal dorsum, 5. The most common duration of effective response was about 4 months, but in eight patients the period was over 5 months. After the response, complete recovery took about 1 or 2 months. Two patients felt unsatisfied, 5 patients felt slightly improved, and 25 patients retained only a slight line and were satisfied with the results. None of the patients experienced complete removal of wrinkle lines. Adverse effects included altered facial looks or appearances, mild local swelling, and ecchymosis at the injection sites. No systemic side effects were noted. Based on these results, the injection of botulinum toxin A seems to be an effective method of eliminating wrinkle lines on the upper third of the face in Korean patients, and it was a simple and effective nonsurgical procedure.