Genes for a nuclease and a protease are involved in the drastic decrease in cellular RNA amount in fission yeast cells during nitrogen starvation

Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation. Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S. pombe undergoing nitrogen starvation. The enzyme was a nuclease encoded by pnu1(+). In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta). During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not. The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease. These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity. The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation. A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.     

Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be k(on) = 32 microm(-1) s(-1) and k(off) = 23 s(-1), respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 degrees C was 0.06 h(-1), which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the nu(Fe-O(2)) band at a noticeably low frequency (560 cm(-1)), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the nu(Fe-His) band at a higher frequency (225 cm(-1)) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the nu(Fe-CO) and nu(C-O) bands at 494 and 1964 cm(-1), respectively, which fall on the same nu(C-O) versus nu(Fe-CO) correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.     

Novel alpha-glucosidase inhibitors with a tetrachlorophthalimide skeleton

Novel alpha-glucosidase inhibitors with a tetrachlorophthalimide skeleton were prepared and their structure-activity relationships were analyzed. Among them, N-phenyl-4,5,6,7-tetrachlorophthalimide (CPOP: 2) and N-(4-phenylbutyl)-4,5,6,7-tetrachlorophthalimide (CP4P: 6) showed very potent inhibitory activity, being more potent than 1-deoxynojirimycin (dNM: 1). Mechanistic studies revealed that CPOP (2) and CP4P (6) inhibit alpha-glucosidase non-competitively and competitively, respectively.     

Daisyworld inhabited with daisies incorporating a seed size/number trade-off: the mechanism of negative feedback on selection from a standpoint of the competition theory

We reexamined a Daisyworld model from the traditional view of competition theory. Unlike the original model, white and black daisies in our model incorporate a seeding/germination trade-off against bare ground area without assuming the local temperature reward. As a result, the planetary temperature is automatically regulated by two species if the following conditions are met: (i) the species react equally to an environmental condition, but one can alter the environmental condition in the opposite direction to the other. (ii) that one of the two cannot have both a higher maximal growth rate (mu(max)) and lower half-saturation constant (K) than those of the other. In other words, a pair of phenotypes incorporates a trade-off between quality and number of seeds. We found that the homeostatic regulation can also be reconciled with the adaptive evolution of optimal temperature. The results of simulation imply that biotic environmental feedback can also be maintained when the emergence of polymorphisms (black and white daisies) is closely linked to such a trade-off.     

Inhibition of human papillomavirus type 16 E7 phosphorylation by the S100 MRP-8/14 protein complex

The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.     

Effect of nitric oxide on gamma-ray-induced micronucleus frequency in RAW264.7 cells

The effect of low-dose nitric oxide (NO) on gamma-ray-induced micronucleus (MN) frequency was investigated in RAW264.7 cells. Treatment of RAW264.7 cells with 0.25 mM sodium nitroprusside (SNP), a chemical NO donor, reduced the frequency of micronuclei induced by 5 Gy gamma rays by 43 to 45% between 3 and 12 h post-treatment. This effect was blocked by carboxy-PTIO, suggesting that NO may play a role in the reduction of radiation-induced MN frequency. To examine possible mechanisms underlying this effect, we first looked at changes in the antioxidant system after SNP treatment. A significant increase in intracellular glutathione (GSH) was seen in SNP-treated cells between 3 and 12 h post-treatment. Depletion of GSH with buthionine sulfoximine (BSO) increased the gamma-ray-induced increase in MN frequency. Detailed studies using various inducers of intracellular GSH suggested that GSH induction has a partial role in the reducing effect of NO on the gamma-ray-induced MN frequency. Next, the effect of NO on DNA repair and replication systems was examined. Wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK), dose-dependently inhibited the reducing effect of NO, while caffeine, an inhibitor of ATM kinase and ATR kinase, did not. DNA-PK activity was increased by NO treatment. Etoposide, a topoisomerase II inhibitor, dose-dependently blocked the effect of NO in reducing the gamma-ray-induced MN frequency. These results suggest that the mechanisms of the effect of NO on the gamma-ray-induced MN frequency include elevation of GSH and up-regulation of DNA-PK activity for repairing double-strand breaks. NO may act as a signal for repair systems, e.g. for nonhomologous recombination and for the replication system in S phase, to reduce the MN frequency.     

Erythromycin differentially inhibits lipopolysaccharide- or poly(I:C)-induced but not peptidoglycan-induced activation of human monocyte-derived dendritic cells

Erythromycin (EM) has attracted attention because of its anti-inflammatory effect. Because dendritic cells (DCs) are the most potent APCs involved in numerous pathologic processes including innate immunity, we examined effects of EM on the activation of human DCs by pathogen-derived stimuli. Monocyte-derived DCs were pretreated with EM and subsequently stimulated with peptidoglycan, polyriboinosinic:polyribocytidylic acid (poly(I:C)), or LPS. The activation of DCs was assessed by surface molecule expression and cytokine production. To reveal the signaling pathways affected by EM, TLR expression, NF-kappaB, IFN regulatory factor-3, and AP-1 activation were examined. EM inhibited costimulatory molecule expression and cytokine production that was induced by poly(I:C) and LPS but not by peptidoglycan. EM pretreatment down- and up-regulated mRNA levels of TLR3 and TLR2, respectively, but did not affect that of TLR4. EM suppressed IFN regulatory factor-3 activation and IFN-beta production but not AP-1 activation induced by poly(I:C) and LPS. The inhibitory effect of EM on NF-kappaB activation was observed only in poly(I:C)-stimulated DCs. EM selectively suppressed activation of DCs induced by LPS and poly(I:C) in different ways, suggesting that the immuno-modulating effects of EM depend on the nature of pathogens. These results might explain why EM prevents the virus-induced exacerbation in the chronic inflammatory respiratory diseases and give us the clue to design new drugs to treat these diseases.     

Direct evidence for the involvement of brain-derived neurotrophic factor in the development of a neuropathic pain-like state in mice

Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were completely suppressed by repeated intrathecal (i.t.) injection of a TrkB/Fc chimera protein, which sequesters endogenous brain-derived neurotrophic factor (BDNF). In addition, BDNF heterozygous (+/-) knockout mice exhibited a significant suppression of nerve ligation-induced thermal hyperalgesia and tactile allodynia compared with wild-type mice. After nerve ligation, BDNF-like immunoreactivity on the superficial laminae of the ipsilateral side of the spinal dorsal horn was clearly increased compared with that of the contralateral side. It should be noted that a single i.t. injection of BDNF produced a long-lasting thermal hyperalgesia and tactile allodynia in normal mice, and these responses were abolished by i.t. pre-treatment with either a Trk-dependent tyrosine kinase inhibitor K-252a or a selective protein kinase C (PKC) inhibitor Ro-32-0432. Supporting these findings, we demonstrated here for the first time that the increase in intracellular Ca2+ concentration by application of BDNF in cultured mouse spinal neurons was abolished by pre-treatment with either K-252a or Ro-32-0432. Taken together, these findings suggest that the binding of spinally released BDNF to TrkB by nerve ligation may activate PKC within the spinal cord, resulting in the development of a neuropathic pain-like state in mice.     

Thallium contamination in wild ducks in Japan

Although thallium (Tl) is toxic to both humans and animals, there is little information on contamination in wildlife. In this study, Tl contents in wild ducks in Japan were determined. Contents of Tl in kidney and liver ranged from 0.42 to 119.61 and 0.10 to 33.94 microg/g dry weight, respectively. Significant correlations between Tl contents in kidney and liver were observed for all dabbling ducks except mallard (Anas platyrhynchos); similar correlations were not observed in diving ducks. Variation in Tl content was observed between sampling locations with the highest mean Tl content in the Eurasian wigeon (Anas penelope) collected in Ibaraki Prefecture.