Methylcobalamin induces a long-lasting enhancement of the field potential in rat suprachiasmatic nucleus slices

Optic nerve stimulation has been reported to evoke a field potential (FP) in rat suprachiasmatic nucleus (SCN) slices. Methylcobalamin,δ-(5,6-dymethylbenzimidazoyl)-Co-methyl-cobamide (Me-B₁₂) enhanced this FP and the enhancement lasted more than 1 h after washing out. Maximal enhancement (143.6±9.8%) was achieved at a concentration of 10 μM. By contrast, cyanocobalamin containing CN- instead of CH₃-showed no enhancement of the amplitude in the FP. Me-B₁₂ induced enhancement of FP was strongly blocked by an N-methyl- -aspartate (NMDA) receptor antagonist, -2-amino-5-phosphonovaleric acid (APV). These results indicate that CH₃- in the Me-B₁₂ is required to modulate the FP amplitude and the NMDA receptor is involved in the long-lasting FP enhancement induced by Me-B₁₂. The present results suggest that Me-B₁₂ modifies the photic entrainment of the circadian clock in the suprachiasmatic nucleus via an activation of NMDA receptors.     

Xeroderma pigmentosum group F protein binds to Eg5 and is required for proper mitosis: implications for XP-F and XFE

The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.     

Th1-type immune responses by Toll-like receptor 4 signaling are required for the development of myocarditis in mice with BCG-induced myocarditis

The immunological aspects of autoimmune myocarditis are difficult to understand because of the existence of many infectious agents and animal models suggesting different mechanisms in autoimmune myocarditis. To overcome these difficulties, two strains of mice, C3H/HeN and C3H/HeJ, showing different immune responses to mycobacteria, were immunized with myosin mixed with BCG. The C3H/HeN mice with a wild-type Toll-like receptor 4 (TLR4) showed severe myocarditis, whereas the C3H/HeJ mice with nonfunctional mutated TLR4 did not. CD4(+) cells from both strains of mice exhibited appreciable proliferative responses following myosin stimulation; however, the cytokines from these cells differed between these two strains. The C3H/HeN mice showed T helper (Th)1-type cytokine responses, whereas the expressions of mRNA in C3H/HeJ mice were Th2-type cytokine. When both of these strains of immunized mice were inoculated with a plasmid encoding cDNA of interleukin (IL)-4 or agonistic IL-4, the development of myocarditis was inhibited in C3H/HeN mice. Moreover, C3H/HeJ mice, in which development of myocarditis was not induced by immunization of myosin mixed with BCG, showed myocarditis after injection of IL-4 antagonistic mutant DNA for the induction of Th1-type immune responses. The results suggested that the induction of autoimmune myocarditis by myosin is affected by Th1-type immune responses.     

Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant

Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1–290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1–290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an amino-terminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.     

Multiple Repeat Resections of Intra- and Extrahepatic Recurrencesin Patients Undergoing Initial Hepatectomy for Colorectal CarcinomaMetastases

Recent evidence suggests that single repeat metastasectomy may provide survival benefits for selected patients experiencing hepatic or pulmonary recurrences following initial hepatectomy for colorectal carcinoma metastases. The aim of this retrospective study was to clarify the efficacy of multiple repeat resections of intra- and extrahepatic recurrences following initial hepatectomy. A total of 100 patients underwent curative partial hepatectomy as the initial procedure for colorectal carcinoma metastases. Tumor relapse after initial hepatectomy was seen in 72 patients, of whom 28 underwent 45 repeat metastasectomies of various sites: 18 patients underwent a single repeat metastasectomy, and 10 underwent multiple repeat metastasectomies. The overall survival rate at 5 years after initial hepatectomy was 36.6%, while the 5-year survival rate after repeat metastasectomy in the 28 patients was 43.6%. The outcome of initial hepatectomy was comparable with that of repeat metastasectomy (p = 0.6924). Among the 28 patients undergoing repeat metastasectomy, the outcome of resection of intrahepatic recurrences in 11 patients was comparable with the outcome of resection of extrahepatic recurrences in 17 patients(p = 0.3926). The outcome of multiple repeat metastasectomies compared favorably with single repeat metastasectomy(p = 0.1803). Multivariate analysis(p < 0.0001) showed that repeat metastasectomy was the strongest prognostic factor. In conclusion, both single and multiple repeat resections of intra- and extrahepatic recurrences after initial hepatectomy are efficacious in colorectal carcinoma patients.Repeat resection should be considered for any resectable recurrences after hepatectomy.     

Time-dependent effect of glutamate on long-term potentiation in the suprachiasmatic nucleus of rats

The effect of glutamate on optic nerve stimulation-evoked field potentials in rat suprachiasmatic nucleus (SCN) was examined in vitro. Glutamate application for 20 min induced long-term potentiation (LTP) of the field potentials in the SCN at nighttime, whereas that induced a weak LTP at daytime. On the other hand, application for 40 min induced LTP in the SCN during the daytime, whereas it induced a weak one at nighttime. These results indicate that the effect of glutamate is dependent on the application time and that the effect is influenced by the duration of glutamate exposure.     

Synthesis of a novel series of benzocycloalkene derivatives as melatonin receptor agonists

We synthesized a novel series of benzocycloalkene derivatives and evaluated their binding affinities to melatonin receptors. To control the spatial position of the amide group, one of the important pharmacophores, we incorporated an endo double bond, an exo double bond (E- and Z-configurations), and a chiral center (R- and S-configurations) at position 1. The indan derivatives with the S-configuration at position 1 were the most promising in terms of potency and selectivity for the human melatonin receptor (MT(1) site), while compounds with the R-configuration showed little potential. Our next attempt was to investigate the most favorable conformation of the methoxy group, the other important pharmacophore for binding to the MT(1) receptor. The introduction of a methyl group at position 5 of the indene ring conserved affinity; however, at position 7, it caused a decrease in affinity. These results suggested that the substitution at position 7 forced the methoxy group to adopt an unfavorable orientation. The optimization of the condensed ring size and substituents led to (S)-8d [(S)-N-[2-(2,3-dihydro-6-methoxy-1H-inden-1-yl)ethyl]propionamide], which had high affinity for the human MT(1) receptor (K(i) = 0.041 nM) but no significant affinity for the hamster MT(3)receptor (K(i) = 3570 nM). In addition, a practical synthetic method of chiral N-[2-(2,3-dihydro-1H-inden-1-yl)ethyl]alkanamides employing asymmetric hydrogenation with (S)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl-Ru has been established.