Genetic variations in humans associated with differences in the course of hepatitis C

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.     

Oxidative modification of proteasome: identification of an oxidation-sensitive subunit in 26 S proteasome

Reactive oxygen species (ROS) have the potential to damage cellular components, such as protein, resulting in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. We have previously shown that a prostaglandin D2 metabolite, 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress, J. Biol. Chem. 276, 12076-12083]. In the present study, to elucidate the molecular mechanism underlying the oxidative stress-mediated cell degeneration, we analyzed the protein carbonylation on SH-SY5Y cells when these cells were submitted to an endogenous inducer of ROS production. Upon exposure of SH-SY5Y cells to this endogenous electrophile, we observed significant accumulation of protein carbonyls within the cells. Proteomic analysis of oxidation-sensitive proteins showed that the major intracellular target of protein carbonylation was one of the regulatory subunits in 26 S proteasome, S6 ATPase. Accompanied by a dramatic increase in protein carbonyls within S6 ATPase, the electrophile-induced oxidative stress exerted a significant decrease in the S6 ATPase activities and a decreased ability of the 26 S proteasome to degrade substrates. Moreover, in vitro oxidation of 26 S proteasome with a metal-catalyzed oxidation system also confirmed that S6 ATPase represents the most oxidation-sensitive subunit in the proteasome. These and the observation that down-regulation of S6 ATPase by RNA interference resulted in the enhanced accumulation of ubiquitinated proteins suggest that S6 ATPase is a molecular target of ROS under conditions of electrophile-induced oxidative stress and that oxidative modification of this regulatory subunit of proteasome may be functionally associated with the altered recognition and degradation of proteasomal substrates in the cells.     

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Settlement of Planulae of the Moon Jellyfish Aurelia aurita onto Hydrophilic Polycarbonate Plates Modified by Atmospheric Plasma Treatment

It has been reported that planula larvae of some jellyfish prefer artificial substrates for settlement. This research focused on the relationship between the settlement of planulae and the wettability of artificial substrate surfaces. We used atmospheric plasmas to change the wettability of the surfaces of polycarbonate (PC) plates because plasma treatment has no chemical side effects. The treatment made the surfaces hydrophilic, as evidenced by the decrease of contact angle from 85° to 35°. X-ray photoelectron spectroscopy revealed that the change of wettability of the PC plates could be attributed to N₂, which was probably ionized in the air above the plates. Scanning electron microscopy revealed no difference in the surface morphology of the plates before and after plasma treatment. Results of bioassays using treated PC plates showed that planulae tended to preferentially settle on hydrophobic surfaces.     

Procedure for the efficient acquisition of progeny seeds from crossed potato plants grafted onto tomato

Potato, Solanum tuberosum L. is an important crop. However, it is difficult to breed potato cultivars by applying conventional crossing methods because potato has a tetraploid genome and is vegetatively propagated. Flower formation and tuber development occur simultaneously. Many potato cultivars hardly produce any fruits after crossing and fail to produce seeds. We report an improved procedure for obtaining progeny seeds by grafting potatoes onto tomatoes. The rate of fruit formation was more than 19% when the grafted potatoes were used for the crossing experiments, whereas crossing using the ungrafted plants showed a rate of 1.1%. This result suggests that our procedure results in the easy acquisition of null-segregant progenies by crossing mutant lines. It is also expected to improve conventional potato breeding.     

Hypoxia-induced fibre type transformation in rat hindlimb muscles Histochemical and electro-mechanical changes

Twelve male Sprague-Dawley rats (21 days old) were randomly assigned into two experimental groups: sea level control (CONT) and hypobaric hypoxia (HYPO). The HYPO rats were kept in an hypobaric chamber maintaining a simulated altitude of 4000 m (61.1 kPa). After 10 weeks of treatment, the rat hindlimb muscles [soleus (SOL) and extensor digitorum longus (EDL)] were subjected to histochemical and electro-mechanical analyses. Results indicated that compared to CONT the HYPO SOL muscle had a significantly greater relative distribution of fast-twitch-oxidative-glycolytic (FOG) fibres (28.9% SEM 2.0 vs 18.3% SEM 1.8, P less than 0.01) with a significant decrease in slow twitch oxidative fibre distribution (69.5% SEM 2.4 vs 82.9% SEM 3.1, P less than 0.01). Compared to CONT the HYPO EDL muscle also manifested a significant increase in FOG fibre distribution (51.6% SEM 0.8 vs 46.6% SEM 1.1, P less than 0.01), but this was accompanied by a significant decrease in fast twitch glucolytic fibres (44.3% SEM 0.9 vs 49.2% SEM 1.7, P less than 0.05). These histochemical fibre type transformations accompanied significant and expected changes in the electro-mechanical parameters tested in situ, e.g. maximal twitch force, maximal rate of force development, contraction time, half relaxation time, force: frequency curve, and fatigability. It was concluded that chronic hypobaric hypoxia could have a potent influence upon the phenotype expression of muscle fibres.     

Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase/oxygenase and the relationship to photosynthetic gas exchange

The kinetic parameters of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) were determined by rapidly assaying the leaf extracts. The respective K _m and V _max values for carboxylase and oxygenase activities were significantly higher for wheat than for rice. In particular, the differences in the V _max values between the two species were greater. When the net activity of CO₂ exchange was calculated at the physiological CO₂-O₂ concentration from these kinetic parameters, it was 22% greater in wheat than in rice. This difference in the in-vitro RuBP-carboxylase/oxygenase activity between the two species reflected a difference in the CO₂-assimilation rate per unit of RuBP-carboxylase protein. However, there was no apparent difference in the CO₂-assimilation rate for a given leaf-nitrogen content between the two species. When the RuBP-carboxylase/oxygenase activity was estimated at the intercellular CO₂ pressure from the enzyme content and kinetic parameters, these estimated enzyme activities in wheat and rice were similar to each other for the same rate of CO₂ assimilation. These results indicate that the difference in the kinetic parameters of RuBP carboxylase between the two species was offset by the differences in RuBP-carboxylase content and conductance for a given leaf-nitrogen content.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic - PAR photosynthetically active radiation - RuBP ribulose-1,5-bisphosphate     

Skewed reproductive success among male white-spotted charr land-locked by an erosion control dam: Implications for effective population size

In 1996 and 1997, the spawning behavior of fluvial white-spotted charr Salvelinus leucomaenis, was observed in the upstream area of an erosion control dam. A small number of males with relatively large body size mated successfully with females as a pair, while almost all satellite males did not sneak successfully, resulting in a non-random mating system. The low sneaking success of subordinate males, in addition to the monopolization of spawning opportunities by a few dominant males, is one of the most important causes of skewed reproductive success among males. The total number of adult fishes in the study area (N: approximately half of the whole tributary above a dam) was estimated as 148 and 102 in 1996 and 1997, respectively. Based on these findings and some further assumptions, the estimated effective population size (N_e) was low in both years. The N_e/N ratio ranged from 0.33 to 0.36 in both years. In addition to reduced population size by construction of an impassable dam, the above-dam population suffered low N_e due to skewed reproductive success among males. The low N_e may be one cause of extinction in above-dam populations of fluvial charr, especially just after the construction of impassable barriers.     

Hepatic oxidative DNA damage correlates with iron overload in chronic hepatitis C patients

Hepatic oxidative stress occurs in chronic hepatitis C (CH-C), but little is known about its producing mechanisms and precise role in the pathogenesis of the disease. To determine the relevance of hepatic oxidatively generated DNA damage in CH-C, 8-hydroxy-2'-deoxyguanosine (8-OHdG) adducts were quantified in liver biopsy specimens by immunohistochemical staining, and its relationship with clinical, biochemical, and histological parameters, and treatment response was assessed in 40 CH-C patients. Hepatic 8-OHdG counts were significantly correlated with serum transaminase levels (r=0.560, p=0.0005) and histological grading activity (p=0.0013). Remarkably, 8-OHdG levels were also significantly related to body and hepatic iron storage markers (vs serum ferritin, r=0.565, p=0.0004; vs hepatic total iron score, r=0.403, p=0.0119; vs hepatic hepcidin messenger RNA, r=0.516, p=0.0013). Baseline hepatic oxidative stress was more prominent in nonsustained virological responder (non-SVR) than in SVR to interferon (IFN)/ribavirin treatment (50.8 vs 32.7 cells/10(5) microm2, p=0.0086). After phlebotomy, hepatic 8-OHdG levels were significantly reduced from 53.4 to 21.1 cells/10(5) microm2 (p=0.0125) with concomitant reductions of serum transaminase and iron-related markers in CH-C patients. In conclusion, this study showed that hepatic oxidatively generated DNA damage frequently occurs and is strongly associated with increased iron deposition and hepatic inflammation in CH-C patients, suggesting that iron overload is an important mediator of hepatic oxidative stress and disease progression in chronic HCV infection.