Genetic variation of NBS-LRR class resistance genes in rice lines

The use of plant disease resistance (R) genes in breeding programs needs an understanding of their variation patterns. In our current study, we investigated the polymorphisms of 44 NBS-LRR class R-genes among 21 rice cultivars and 14 wild rice populations. Our data suggested that there were four basic types of variations: conserved, diversified, intermediate-diversified, and present/absent patterns. Common characteristics at a locus of conserved R-genes were: copy-number uniformity, clear divergence (long branches) with other paralogs, and highly identical alleles. On the other hand, copy-number variability, a nearly equal and non-zero branch lengths, and high levels of nucleotide diversity were observed at the loci of highly diversified R-genes. Research suggests that the ratio of diverse alleles to the total number of genes at a locus is one of the best criteria to characterize the variation pattern of an R-gene. Our data suggested that a significant genetic reduction was detected only in four present/absent R-genes, compared with the variation observed in wild rice. In general, no difference was detected between wild rice and cultivars, japonica and indica rice, or between lines from different geographic regions. Our results also suggested that R-genes were under strong selection, which shaped R-gene variation patterns. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sihai Yang and Tingting Gu contributed equally to this work.     

Chk2-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repair

The DNA damage response (DDR) has an essential function in maintaining genomic stability. Ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2) and ATM- and Rad3-related (ATR)-Chk1, triggered, respectively, by DNA double-strand breaks and blocked replication forks, are two major DDRs processing structurally complicated DNA damage. In contrast, damage repaired by base excision repair (BER) is structurally simple, but whether, and how, the DDR is involved in repairing this damage is unclear. Here, we demonstrated that ATM-Chk2 was activated in the early response to oxidative and alkylation damage, known to be repaired by BER. Furthermore, Chk2 formed a complex with XRCC1, the BER scaffold protein, and phosphorylated XRCC1 in vivo and in vitro at Thr(284). A mutated XRCC1 lacking Thr(284) phosphorylation was linked to increased accumulation of unrepaired BER intermediate, reduced DNA repair capacity, and higher sensitivity to alkylation damage. In addition, a phosphorylation-mimic form of XRCC1 showed increased interaction with glycosylases, but not other BER proteins. Our results are consistent with the phosphorylation of XRCC1 by ATM-Chk2 facilitating recruitment of downstream BER proteins to the initial damage recognition/excision step to promote BER.     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.     

Expression and characterization of two secreted His6-tagged endo-beta-1,4-glucanases from the mollusc Ampullaria crossean in Pichia pastoris

Two endo-β-1,4-glucanase cDNAs, eg27I and eg27II , from the mollusc Ampullaria crossean were expressed in Pichia pastoris cells. The secreted His₆-tagged proteins were purified in a single chromatography step. The purified recombinant EG27I and EG27II showed enzymatic activity on carboxylmethyl cellulose sodium salt at 15.31 U/mg and 12.40 U/mg, respectively. The optimum pH levels of the recombinant EG27I and EG27II were 5.5 and 5.5–6.0, respectively, and the optimum temperatures were 50°C and 50°C–55°C, respectively. The pH stability study revealed that both EG27I and EG27II showed their highest stability at pH 8.0. Analysis of their thermostability indicated that both EG27I and EG27II were relatively stable up to 40°C. Site-directed mutagenesis of Asp43 and Asp153 of both EG27I and EG27II showed that the two Asp residues are critical for the enzymatic activity.     

Effects of special brain area regional cerebral blood flow abnormal perfusion on learning and memory function and its molecular mechanism in rats

To study the effect of special brain area regional cerebral blood flow (rCBF) abnormal perfusion on learning and memory function and its molecular mechanism, 64 adult male healthy Sprague-Dawley (SD) rats were randomly divided into two groups, the false operation group (control group) and the operation group (model group). After surgical operation, the operation group undertook bilateral common carotid artery permanent ligation, while the other group did not. Learning and memory function were measured by Y-maze at 4 h, 8 h, 24 h and 3 d after surgical operation, respectively. The rCBF of the right frontal lobe and hippocampus was also detected by the PerifluxPF model laser Doppler flowmetry, and the expressions of c-fos or c-jun or Bcl-2 and Bax were also measured by immune histochemistry S-P method accordingly. Results showed that the rCBF of the right frontal lobe and hippocampus in the operation group was significantly lower than that in the false operation group (P < 0.05). The learning indexes, error number (EN), day of reach standard and total reaction time (TRT) in the operation group, were significantly higher than that in the false operation group (P < 0.05). However, the initiative evasion rate in the operation group was significantly lower than that in the false operation group. The study also found that the rCBF was relatively more, the indexes (EN, the day of reach standard and TRT) relatively fewer, but the initiative evasion rate and the memory keeping rate were relatively more. The positive expression and the average absorbency of Fos and Jun in the operation group were significantly higher than that in the false operation group (P < 0.05). Furthermore, Bax and Bcl-2 positive cells were all increased over time in the operation group, and the expression ratio of Bax/Bcl-2 in the operation group was significantly higher than that in the false operation group (P < 0.01). In conclusion, rCBF decrease can impair the learning and memory function in rats, which may be related to the increase of the expression ratio of c-fos or c-jun or Bcl-2 or Bax in the frontal cortex and hippocampus.     

SnoRNA Snord116 (Pwcr1/MBII-85) deletion causes growth deficiency and hyperphagia in mice

Prader-Willi syndrome (PWS) is the leading genetic cause of obesity. After initial severe hypotonia, PWS children become hyperphagic and morbidly obese, if intake is not restricted. Short stature with abnormal growth hormone secretion, hypogonadism, cognitive impairment, anxiety and behavior problems are other features. PWS is caused by lack of expression of imprinted genes in a approximately 4 mb region of chromosome band 15q11.2. Our previous translocation studies predicted a major role for the C/D box small nucleolar RNA cluster SNORD116 (PWCR1/HBII-85) in PWS. To test this hypothesis, we created a approximately 150 kb deletion of the > 40 copies of Snord116 (Pwcr1/MBII-85) in C57BL/6 mice. Snord116del mice with paternally derived deletion lack expression of this snoRNA. They have early-onset postnatal growth deficiency, but normal fertility and lifespan. While pituitary structure and somatotrophs are normal, liver Igf1 mRNA is decreased. In cognitive and behavior tests, Snord116del mice are deficient in motor learning and have increased anxiety. Around three months of age, they develop hyperphagia, but stay lean on regular and high-fat diet. On reduced caloric intake, Snord116del mice maintain their weight better than wild-type littermates, excluding increased energy requirement as a cause of hyperphagia. Normal compensatory feeding after fasting, and ability to maintain body temperature in the cold indicate normal energy homeostasis regulation. Metabolic chamber studies reveal that Snord116del mice maintain energy homeostasis by altered fuel usage. Prolonged mealtime and increased circulating ghrelin indicate a defect in meal termination mechanism. Snord116del mice, the first snoRNA deletion animal model, reveal a novel role for a non-coding RNA in growth and feeding regulation.     

Tree of life based on genome context networks

Efforts in phylogenomics have greatly improved our understanding of the backbone tree of life. However, due to the systematic error in sequence data, a sequence-based phylogenomic approach leads to well-resolved but statistically significant incongruence. Thus, independent test of current phylogenetic knowledge is required. Here, we have devised a distance-based strategy to reconstruct a highly resolved backbone tree of life, on the basis of the genome context networks of 195 fully sequenced representative species. Along with strongly supporting the monophylies of three superkingdoms and most taxonomic sub-divisions, the derived tree also suggests some intriguing results, such as high G+C gram positive origin of Bacteria, classification of Symbiobacterium thermophilum and Alcanivorax borkumensis in Firmicutes. Furthermore, simulation analyses indicate that addition of more gene relationships with high accuracy can greatly improve the resolution of the phylogenetic tree. Our results demonstrate the feasibility of the reconstruction of highly resolved phylogenetic tree with extensible gene networks across all three domains of life. This strategy also implies that the relationships between the genes (gene network) can define what kind of species it is.     

Functional role of BLAP75 in BLM-topoisomerase IIIalpha-dependent holliday junction processing

The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo) IIIalpha to form an evolutionarily conserved complex, termed the BTB complex, that functions to regulate homologous recombination. BLAP75 binds DNA, associates with both BLM and Topo IIIalpha, and enhances the ability of the BLM-Topo IIIalpha pair to branch migrate the Holliday junction (HJ) or dissolve the double Holliday junction (dHJ) structure to yield non-crossover recombinants. Here we seek to understand the relevance of the biochemical attributes of BLAP75 in HJ processing. With the use of a series of BLAP75 protein fragments, we show that the evolutionarily conserved N-terminal third of BLAP75 mediates complex formation with BLM and Topo IIIalpha and that the DNA binding activity resides in the C-terminal third of this novel protein. Interestingly, the N-terminal third of BLAP75 is just as adept as the full-length protein in the promotion of dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. Thus, the BLAP75 DNA binding activity is dispensable for the ability of the BTB complex to process the HJ in vitro. Lastly, we show that a BLAP75 point mutant (K166A), defective in Topo IIIalpha interaction, is unable to promote dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. This result provides proof that the functional integrity of the BTB complex is contingent upon the interaction of BLAP75 with Topo IIIalpha.     

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF)

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the ⁶CPCP⁹ motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.     

Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines

Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.