ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue

Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3′ end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3′ end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding.     

Contribution of Nucleus Accumbens Core (AcbC) to Behavior Control during a Learned Resting Period: Introduction of a Novel Task and Lesion Experiments

In recent years, the study of resting state neural activity has received much attention. To better understand the roles of different brain regions in the regulation of behavioral activity in an arousing or a resting period, we developed a novel behavioral paradigm (8-arm food-foraging task; 8-arm FFT) using the radial 8-arm maze and examined how AcbC lesions affect behavioral execution and learning. Repetitive training on the 8-arm FFT facilitated motivation of normal rats to run quickly to the arm tips and to the center platform before the last-reward collection. Importantly, just after this point and before confirmation of no reward at the next arm traverse, locomotor activity decreased. This indicates that well-trained rats can predict the absence of the reward at the end of food seeking and then start another behavior, namely planned resting. Lesions of the AcbC after training selectively impaired this reduction of locomotor activity after the last-reward collection without changing activity levels before the last-reward collection. Analysis of arm-selection patterns in the lesioned animals suggests little influence of the lesion in the ability to predict the reward absence. AcbC lesions did not change exploratory locomotor activity in an open-field test in which there were no rewards. This suggests that the AcbC controls the activity level of planned resting behavior shaped by the 8-arm FFT. Rats receiving training after AcbC lesioning showed a reduction in motivation for reward seeking. Thus, the AcbC also plays important roles not only in controlling the activity level after the last-reward collection but also in motivational learning for setting the activity level of reward-seeking behavior.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997     

Toll-like Receptors as a Target of Food-derived Anti-inflammatory Compounds

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.     

Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography

14 microsomal cytochromes P-450 were purified from the liver of untreated and phenobarbital- or 3-methylcholanthrene-treated male rats. Following solubilization of microsomes with sodium cholate, poly(ethylene glycol) fractionation and aminohexyl-Sepharose 4B chromatography, cytochromes P-450 were purified by high-performance liquid chromatography (HPLC), using a preparative DEAE-anion-exchange column. The pass-through fraction was further purified by HPLC using a cation-exchange column. Other fractions eluted on preparative DEAE-HPLC were further applied onto an HPLC using a DEAE-column. Five kinds (P-450UT-2-6), four kinds (P-450PB-1,2,4 and 5) and five kinds (P-450MC-1-5) of cytochromes P-450 were purified from untreated rats or rats treated with phenobarbital or 3-methylcholanthrene, respectively. HPLC profiles of tryptic peptides of cytochromes P-450UT-2 and P-450MC-2 were identical and the other profiles obtained from seven purified cytochromes P-450 were distinct from each other. Amino-terminal sequences of eight forms of cytochrome P-450 (UT-2, UT-5, PB-1, PB-2, PB-4, PB-5, MC-1 and MC-5) were distinct except for cytochromes P-450PB-4 and P-450PB-5.     

Characterization of three strains of Capnocytophaga canis isolated from patients with sepsis

Capnocytophaga canimorsus and Capnocytophaga cynodegmi, both commensal bacteria in the oral cavities of dogs and cats, are zoonotic pathogens. In particular, C. canimorsus causes sepsis and fatal septic shock. Recently, a novel Capnocytophaga species, C. canis, was isolated from the oral cavities of healthy dogs. It is reportedly oxidase‐negative and therefore considered avirulent in humans. In the present study, three strains of C. canis were isolated from Japanese patients with sepsis. All three strains, HP20001, HP33001 and HP40001, were oxidase‐positive. Nucleotide sequence identities of the 16S rRNA gene of the three strains to the C. canimorsus type strain ATCC35979, C. cynodegmi type strain ATCC49044 and C. canis type strain LMG29146 were 96.9–97.0%, 96.9–97.0% and 99.7–99.8%, respectively. Multi‐locus sequence analysis based on seven house‐keeping genes, dnaJ, fumC, glyA, gyrB, murG, trpB and tuf, revealed that the oxidase‐positive C. canis strains isolated in Japan and oxidase‐negative strains of C. canis from canine oral cavities in Switzerland were clustered in different genetic subgroups. These results indicate that the virulence of C. canis strains in humans is associated with oxidase activity.

     

Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycine max (L.) Merr.]

Crop leaves are subject to continually changing light levels in the field. Photosynthetic efficiency of a crop canopy and productivity will depend significantly on how quickly a leaf can acclimate to a change. One measure of speed of response is the rate of photosynthesis increase toward its steady state on transition from low to high light. This rate was measured for seven genotypes of soybean [Glycine max (L.) Merr.]. After 10 min of illumination, cultivar ‘UA4805’ (UA) had achieved a leaf photosynthetic rate (P_n) of 23.2 μmol · m^?2 · s^?1, close to its steady‐state rate, while the slowest cultivar ‘Tachinagaha’ (Tc) had only reached 13.0 μmol · m^?2 · s^?1 and was still many minutes from obtaining steady state. This difference was further investigated by examining induction at a range of carbon dioxide concentrations. Applying a biochemical model of limitations to photosynthesis to the responses of P_n to intercellular CO₂ concentration (C_i), it was found that the speed of apparent in vivo activation of ribulose‐1:5‐bisphosphate carboxylase/oxygenase (Rubisco) was responsible for this difference. Sequence analysis of the Rubisco activase gene revealed single nucleotide polymorphisms that could relate to this difference. The results show a potential route for selection of cultivars with increased photosynthetic efficiency in fluctuating light.     

Loss of SDHB Elevates Catecholamine Synthesis and Secretion Depending on ROS Production and HIF Stabilization

Germline mutations in genes encoding succinate dehydrogenase subunits are associated with the development of familial pheochromocytomas and paragangliomas [hereditary paraganglioma/pheochromocytoma syndrome (HPPS)]. In particular, a mutation in succinate dehydrogenase subunit B (SDHB) is highly associated with abdominal paraganglioma and subsequent distant metastasis (malignant paraganglioma), indicating the importance of SDHB genetic testing. The discovery of HPPS suggests an association among genetic mitochondrial defects, tumor development, and catecholamine oversecretion. To investigate this association, we transfected pheochromocytoma cells (PC12) with SDHB-specific siRNA. SDHB silencing virtually abolished complex II activity, demonstrating the utility of this in vitro model for investigating the pseudo-hypoxic drive hypothesis. Lack of complex II activity resulting from RNA interference of SDHB increased tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine biosynthesis) activity and catecholamine secretion. Reduced apoptosis was observed accompanied by Bcl-2 accumulation in PC12 cells, consistent with the phenotypes of paragangliomas with SDHB mutations. In addition, SDHB silencing increased reactive oxygen species (ROS) production and nuclear HIF1α stabilization under normoxic conditions. Furthermore, phenotypes induced by complex II activity knockdown were abolished by pretreatment with N-acetyl cysteine (an ROS scavenger) and by prior HIF1α knockdown, indicating an ROS- and HIF1α-dependent mechanism. Our results indicate that increased ROS may act as signal transduction messengers that induce HIF1α stabilization and may be necessary for the pseudo-hypoxic states observed in our experimental model. To our knowledge, this is the first study demonstrating that pseudo-hypoxic states resulting from SDHB knockdown are associated with increased TH activity and catecholamine oversecretion.     

Reconstitution of urea-dissociated subunits of a pig heart phosphoprotein phosphatase

Pig heart phosphoprotein phosphatase [phosphoprotein phosphophydrolase, EC 3.1.3.16] of Mr 224,000 was dissociated by gel-filtration on Sephacryl S-300, into an active subunit (alpha subunit) of Mr 31,000 and inactive subunits of higher molecular weight in the presence of 6 M urea. After the removal of urea, these subunits reassociated, forming two enzyme forms of Mr 237,000 (Form 1) and Mr 123,000 (Form 2). Form 2 was produced by association of the alpha subunit with an inactive subunit (beta subunit) of Mr 80,000, while Form 1 was formed by combination of the alpha subunit with a complex of inactive subunits which was eluted from a Sephadex G-150 column in fractions of molecular weight range greater than 80,000. The dissociation and reassociation of the subunits of Form 1 by the same urea method produced not only Form 1, but also significant amounts of Form 2, indicating that the inactive subunits of Form 1 were a complex of the beta subunit with another inactive subunit(s). The molecular parameters and other properties of Form 1 were very close to those of the original enzyme. By the conversion of Form 1 to Form 2, the activities of Form 1 towards phosphorylase a and glycogen synthetase b were enhanced 2-3 fold with no significant change in activity towards P-H1 histone or in response to the stimulatory effect of Mg(CH3COO)2 on the dephosphorylation of P-H2B histone. However, removal of the beta subunit from From 2 resulted in strong suppression of activity towards P-H1 histone and response to the salt effect with lesser effects on the activities of Form 2 towards phosphorylase a and glycogen synthase b.