High-Performance Breaking and Intelligent of Miniature Circuit Breakers.

The exploitation and utilization of clean energy such as wind and photovoltaic power plays an important role in the reduction in carbon emissions to achieve the goal of "emission peak and carbon neutral", but such a quantity of clean energy accessing the electric system will foster the transition of the electric power system structure. The intelligentization of power equipment will be an inevitable trend of development. High breaking performance, remote control and a digital detection platform of miniature circuit breaker, a protective equipment of a power distribution system, have also been inevitable requirements of the power Iot system. Based on the above, this paper studies three aspects: high-performance AC and DC general switching technology, remote control technology and operation status' digital monitoring. A new DC non-polar breaking technology is proposed, which improves the short circuit breaking ability. An experimental prototype using the above techniques was fabricated and passed the DC 1000 V/10 kA short-circuit breaking test. On the basis of the above, an intelligent circuit breaker is developed, which contains multiple functions: remote switching, real-time temperature detection, energy metering and fault warning. Moreover, a software for digital condition monitoring and remote control is developed. This work has certain theoretical and practical significance for the development of the power Internet of things.

A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of ß-Cell Glucolipotoxicity.

Type 2 diabetes (T2D) is caused by loss of pancreatic ß-cell mass and failure of the remaining ß-cells to deliver sufficient insulin to meet demand. ß-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on ß-cell function and survival, contributes to T2D-associated ß-cell failure. Drugs and mechanisms that protect ß-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected ß-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured ß-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve ß-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

Proteomic changes in rice leaves during development of field-grown rice plants.

Of the numerous factors affecting rice yield, how solar radiation is transformed into biomass through rice leaves is the most important. We have analyzed proteomic changes in rice leaves collected from six different developing stages (vegetative to ripening). We studied protein expression profiles of rice leaves by running two-dimensional gel electrophoresis. Differential protein expression among the six phases were analyzed by image analysis, which allowed the identification of 49 significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which 89.8% of them were confirmed to be rice proteins. Finally, we confirmed some of the interesting rice proteins by immunoblotting. Three major conclusions can be drawn from these experimental results. (i) Protein expression in rice leaves, at least for high or middle abundance proteins, is attenuated during growth (especially some chloroplast proteins). However, the change is slow and the expression profiles are relatively stable during rice development. (ii) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major protein in rice leaves, is expressed at constant levels at different growth stages. Interestingly, a high ratio of degradation of the RuBisCO large subunit was found in all samples. This was confirmed by two approaches, mass spectrometry and immunoblotting. The degraded fragments are similar to other digested products of RuBisCO mediated by free radials. (iii) The expression of antioxidant proteins such as superoxide dismutase and peroxidase decline at the early ripening stage.

The Genomes of Oryza sativa: a history of duplications.

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000-40,000. Only 2%-3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.

Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes.

COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to repress photomorphogenesis. Here, we demonstrate that COP10 interacts with ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in vitro, an activity distinct from previous characterized UEVs such as MMS2 and UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically interacts with COP1 and the CSN. Purified CDD (COP10, DDB1, DET1) complex also shows enhancement of E2 activity (UEA) similar to that observed with COP10 itself. Our data suggests that COP10, along with COP1 and the CSN, promotes the degradation of positive regulators of photomorphogenesis, such as the transcription factor HY5, via the ubiquitin/26S proteasome system. Thus, the CDD complex may act as a ubiquitylation-promoting factor to regulate photomorphogenesis.

Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys.

Snake venom is a complex mixture of proteins and peptides, and a number of studies have described the biological properties of several venomous proteins. Nevertheless, a complete proteomic profile of venom from any of the many species of snake is not available. Proteomics now makes it possible to globally identify proteins from a complex mixture. To assess the venom proteomic profiles from Naja naja atra and Agkistrodon halys, snakes common to southern China, we used a combination strategy, which included the following four different approaches: (i) shotgun digestion plus HPLC with ion-trap tandem MS, (ii) one-dimensional SDS/PAGE plus HPLC with tandem MS, (iii) gel filtration plus HPLC with tandem MS and (iv) gel filtration and 2DE (two-dimensional gel electrophoresis) plus MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) MS. In the present paper, we report the novel identification of 124 and 74 proteins and peptides in cobra and viper venom respectively. Functional analysis based upon toxin categories reveals that, as expected, cobra venom has a high abundance of cardio- and neurotoxins, whereas viper venom contains a significant amount of haemotoxins and metalloproteinases. Although approx. 80% of gel spots from 2DE displayed high-quality MALDI-TOF-MS spectra, only 50% of these spots were confirmed to be venom proteins, which is more than likely to be a result of incomplete protein databases. Interestingly, these data suggest that post-translational modification may be a significant characteristic of venomous proteins.

An analysis of the proteomic profile for Thermoanaerobacter tengcongensis under optimal culture conditions.

The genome of Thermoanaerobacter tengcongensis is estimated to encode 2588 theoretical proteins. In this study, we have vitalized approximately 46% of the theoretical proteome experimentally using a proteomic strategy that combines three different methods, shotgun digestion plus high-performance liquid chromatography (HPLC) with ion-trap tandem mass spectrometry (shotgun-liquid chromatography (LC)/MS), one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus HPLC with ion-trap tandem mass spectrometry (one-dimensional electrophoresis (1DE)-LC/MS), and two-dimensional gel electrophoresis plus matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (2DE-MALDI-TOF-MS). Of the 1200 proteins identified, as few as 76 proteins were globally found by all three approaches, and notably, most of these proteins were in the soluble fraction. However, there were a number of unique proteins detected by one method only, suggesting that our strategy provides a means toward obtaining a comprehensive view of protein expression profile. Proteins from the major metabolic pathways are strongly represented on the map, and a number of these enzymes were identified by more than one proteomic method. Based upon the proteins identified in the present study, we are able to broaden the understanding of how T. tengcongensis survives under high temperature environment, whereas several of its properties can not be fully explained by genome data.

Assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus.

BACKGROUND: The widespread threat of severe acute respiratory syndrome (SARS) to human life has spawned challenges to develop fast and accurate analytical methods for its early diagnosis and to create a safe antiviral vaccine for preventive use. Consequently, we thoroughly investigated the immunoreactivities with patient sera of a series of synthesized peptides from SARS-coronavirus structural proteins. METHODS: We synthesized 41 peptides ranging in size from 16 to 25 amino acid residues of relatively high hydrophilicity. The immunoreactivities of the peptides with SARS patient sera were determined by ELISA. RESULTS: Four epitopic sites, S599, M137, N66, and N371-404, located in the SARS-coronavirus S, M, and N proteins, respectively, were detected by screening synthesized peptides. Notably, N371 and N385, located at the COOH terminus of the N protein, inhibited binding of antibodies to SARS-coronavirus lysate and bound to antibodies in >94% of samples from SARS study patients. N385 had the highest affinity for forming peptide-antibody complexes with SARS serum. CONCLUSIONS: Five peptides from SARS structural proteins, especially two from the COOH terminus of the N protein, appear to be highly immunogenic and may be useful for serologic assays. The identification of these antigenic peptides contributes to the understanding of the immunogenicity and persistence of SARS coronavirus.

Protein kinase C-dependent phosphorylation of the beta-subunit of the voltage-sensitive potassium channels (Kvbeta2).

The K(v)beta proteins are members of the aldo-keto reductase (AKR) superfamily that interact with the cytoplasmic face of the pore-forming alpha-subunits of the voltage-sensitive K(+) channels. The physiological functions of K(v)beta are unclear, although some members of the K(v)beta family impart rapid inactivation to non-inactivating K(+) channels. Structural analyses reveal that the NADPH-binding site as well as the active site architecture and residues of other AKR proteins are conserved in the K(v)beta proteins. The K(v)beta2 displays high-affinity NADPH binding, although no catalytic activity for this protein has been reported. Recent studies show that K(v)beta2 is constitutively associated with protein kinase C (PKC) zeta via the zeta-interacting protein (ZIP). The primary structure of K(v)beta displays 25 PKC consensus phosphorylation sites. We report that incubation of recombinant K(v)beta2 with PKCalpha in vitro leads to rapid phosphorylation of the protein. Stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) also induced the phosphorylation of K(v)beta2 expressed in COS-7 cells. Deletion of the first 35 amino acids of the variable N-terminus led to a substantial decrease in the overall extent of phosphorylation in vitro, indicating that the N-terminus peptide is the preferred site of PKC phosphorylation. Analysis of the phosphorylated protein by HPLC coupled with an ion-trap electrospray mass spectrometer revealed phosphorylation of Ser-266 located near the NADPH-binding site of the protein. We propose that phosphorylation could affect the N-terminus-mediated beta-alpha interactions or the binding of NADP(H) to the conserved AKR domain of the K(v)beta proteins.

A novel approach for identifying the heme-binding proteins from mouse tissues.

Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of heme-binding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by heme-agarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2-DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity.

In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

The epitope study on the SARS-CoV nucleocapsid protein.

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.

A strategy for searching antigenic regions in the SARS-CoV spike protein.

In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA), these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis.

[Apoptosis antagonizing transcription factor is a gene highly expressed in highly metastatic human osteosarcoma cell lines].

BACKGROUND & OBJECTIVE: Osteosarcoma is the most common malignant bone tumor in adolescents and yound adults. It is characterized by a high propensity for pulmonary metastasis. In spite of successful control of the primary tumor, death from pulmonary metastases occurs in > 30% of patients within 5 years. A better understanding of the molecular mechanisms that regulate the process of metastasis can provide a biological foundation for the design of more effective therapy. We established a metastatic model in nude mice with the method of orthotopically transplanting human osteosarcoma cell line SOSP-9607 and selected and isolated SOSP-M with highly metastatic potential. This study is to clone genes associated with osteosarcoma metastasis and to investigate the molecular mechanism of osteosarcoma metastasis by comparing the levels of gene expression between the two cells lines. METHODS: Using suppression subtractive hybridization, the substracted cDNA library of highly metastatic human osteosarcoma cell line SOSP-M was constructed. Positive clones were screened by differential screen technique. Partial positive clones were sequenced. The interested upexpressed clones in SOSP-M cells were analyzed through Northern blot and RT-PCR for the low metastatic cell lines SOSP-9607 and OS-9901, highly metastatic cell line SOSP-M and three pulmonic metastatic nodules. RESULTS: Two positive cDNA clones from highly metastatic cell line SOSP-M subtracted cDNA library were identical(99% homology) to apoptosis antagonizing transcription factor. Northern blot and RT-PCR analysis demonstrated that apoptosis antagonizing transcription factor expressed highly in high metastatic cell line SOSP-M and three pulmonic metastatic nodules, but not in low metastatic cell line SOSP-9607 and OS-9901. CONCLUSION: Apoptosis antagonizing transcription factor may play an important role in promoting metastasis of osteosarcoma.

[Identification of genes associated with human osteosarcoma metastasis suppression using suppression subtractive hybridization].

OBJECTIVE: To identify genes associated with metastasis suppression and to investigate the molecular mechanism of osteosarcoma metastasis. METHODS: The subtracted cDNA library of low metastatic human osteosarcoma cell line SOSP-9607 was constructed using suppression subtractive hybridization. Partial clones were sequenced. The acquired sequence data were aligned against the GenBank nucleotide database using Blastn to search for sequence matches. The interested clone was used to perform Northern blot and reverse transcriptase-PCR (RT-PCR) analysis on mRNA isolated from low metastatic cell line SOSP-9607 and OS-9901, high metastatic cell line SOSP-M and three pulmonic metastatic nodules of nude mice. RESULTS: A cDNA clone from low metastatic cell line SOSP-9607 subtracted cDNA library was identified as telomeric repeat binding factor 2 (TERF2) by sequence analysis and Blastn search. Northern blot and RT-PCR analysis demonstrated that TERF2 expressed highly in low metastatic cell line SOSP-9607 and OS-9901, but not in high metastatic cell line SOSP-M and three pulmonic metastatic nodules. CONCLUSION: TERF2 may be important for suppressing metastasis of osteosarcoma.