The rosettazyme: A synthetic cellulosome

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds.Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg²⁺ assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin–rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.     

Polymorphism attribution of cSNPs in cancer-related genes located in loss regions with a high frequency of HCC between HBV and health groups

Cancer-related genes harbored in the loss regions containing a high frequency of hepatocellular carcinoma (HCC) were selected.Related information was gathered and the coding single nucleotide polymorphism (cSNP) sequences were obtained from the single nucleotide polymorphism (SNP) database.The appropriate primers and oligonucleotide probes were then designed in accordance with the SNP sites,and subsequently,the gene chips for detecting SNPs were constructed.Genomic DNA was extracted from blood samples of healthy controls and from patients with HBV infection.The sequences,including the SNPs,were amplified via polymerase chain reaction (PCR) and labeled using digoxigenin deoxyuridine tri-phosphate (Dig-dUTP).The labeled products were then hybridized with the SNP chips.Results confirmed that the differences in allele frequencies of three SNPs EGFL3 (rs947345),Caspase9 (rs2308950),and E2F2 (rs3218171) were distinct between HBV-infected patients and controls,suggesting that these SNPs ocuring in high frequency in HBV-infected individuals may be associated with susceptibility to HCC.     

Impaired pulmonary artery contractile responses in a rat model of microgravity: role of nitric oxide.

Vascular contractile hyporesponsiveness is an important mechanism underlying orthostatic intolerance after microgravity. Baroreceptor reflexes can modulate both pulmonary resistance and capacitance function and thus cardiac output. We hypothesized, therefore, that pulmonary vasoreactivity is impaired in the hindlimb-unweighted (HLU) rat model of microgravity. Pulmonary artery (PA) contractile responses to phenylephrine (PE) and U-46619 (U4) were significantly decreased in the PAs from HLU vs. control (C) animals. N(G)-nitro-L-arginine methyl ester (10(-5) M) enhanced the contractile responses in the PA rings from both C and HLU animals and completely abolished the differential responses to PE and U4 in HLU vs. C animals. Vasorelaxant responses to ACh were significantly enhanced in PA rings from HLU rats compared with C. Moreover, vasorelaxant responses to sodium nitroprusside were also significantly enhanced. Endothelial nitric oxide synthase (eNOS) and soluble guanlyl cyclase expression were significantly enhanced in PA and lung tissue from HLU rats. In marked contrast, the expression of inducible nitric oxide synthase was unchanged in lung tissue. These data support the hypothesis that vascular contractile responsiveness is attenuated in PAs from HLU rats and that this hyporesponsiveness is due at least in part to increased nitric oxide synthase activity resulting from enhanced eNOS expression. These findings may have important implications for blood volume distribution and attenuated stroke volume responses to orthostatic stress after microgravity exposure.     

A mutant Arabidopsis heterotrimeric G-protein beta subunit affects leaf, flower, and fruit development.

A genetic screen was performed to find new mutants with an erecta (er) phenotype and to identify genes that may function with ER, a receptor-like kinase. These mutants were named elk (for erecta-like) and were placed into five complementation groups. We positionally cloned ELK4 and determined that it encodes AGB1, a putative heterotrimeric G-protein beta subunit. Therefore, elk4 was renamed agb1. agb1-1 plants express similar fruit phenotypes, as seen in er plants, but differ from er in that the stem is only slightly shorter than that in the wild type, the pedicel is slightly longer than that in the wild type, and the leaves are rounder than those in er mutants. Molecular analysis of agb1-1 indicates that it is likely a null allele. AGB1 mRNA is expressed in all tissues tested but is highest in the silique. Analysis of agb1-1 er double mutants suggests that AGB1 may function in an ER developmental pathway regulating silique width but that it functions in parallel pathways affecting silique length as well as leaf and stem development. The finding that AGB1 is involved in the control of organ shape suggests that heterotrimeric G-protein signaling is a developmental regulator in Arabidopsis.     

Purification and partial sequencing of saxiphilin, a saxitoxin-binding protein from the bullfrog, reveals homology to transferrin

Plasma from the bullfrog, Rana catesbeiana, contains a soluble component of unknown function that specifically binds the neurotoxin, [3H]saxitoxin, with a Kd of approximately 0.2 nM. Saxiphilin, the protein responsible for this activity, was purified approximately 440-fold from bullfrog plasma by column chromatography on heparin-Sepharose followed by chromatofocusing. The purified saxiphilin preparation exhibits a binding capacity of 9.6 nmol/mg protein and a Kd of 0.32 nM for [3H]saxitoxin. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a predominant band migrating with an apparent Mr of approximately 89,000 which is similar to the expected size of saxiphilin previously estimated by nondenaturing size exclusion chromatography. Amino-terminal sequencing of the approximately 89-kDa protein and sequencing of four different tryptic peptide fragments revealed that each of the partial saxiphilin sequences can be aligned by homology with members of the transferrin protein family with sequence identity as high as 69%. The available sequence corresponding to conserved residues that comprise part of the two Fe3+ binding sites in lacto-transferrin show several substitutions in saxiphilin, suggesting that saxiphilin is not an Fe(3+)-binding protein. Saxiphilin appears to be a monomeric approximately 89-kDa protein that is evolutionarily related to transferrin but which binds saxitoxin instead of Fe3+.     

Enantioselective Synthesis and Antimicrobial Activities of Tetrahydro‐Β‐Carboline Diketopiperazines

A series of single isomers tetrahydro‐β‐carboline diketopiperazines were stereoselectively synthesized starting from l ‐tryptophan methyl ester hydrochloride and six aldehydes through a four‐step reaction including Pictet‐Spengler reaction, crystallization‐induced asymmetric transformations (CIAT), Schotten‐Baumann reaction, and intramolecular ester amidation. The chemical structures were characterized by nuclear magnetic resonance (NMR) and elemental analysis, among which two compounds were determined by x‐ray single crystal diffraction. Moreover, antimicrobial activities of all the compounds were also tested. Chirality 25:656–662, 2013. © 2013 Wiley Periodicals, Inc.     

Identification and Validation of a Prognostic Model Based on Three MVI-Related Genes in Hepatocellular Carcinoma

MVI has significant clinical value for treatment selection and prognosis evaluation in hepatocellular carcinoma (HCC). We aimed to construct a model based on MVI-Related Genes (MVIRGs) for risk assessment and prognosis prediction in patients with HCC. This study utilized various statistical analysis methods for prognostic model construction and validation in the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts, respectively. In addition, immunohistochemistry and qRT-PCR were used to analyze and identify the value of the model in our cohort. After the analyses, 153 differentially expressed MVIRGs were identified, and three key genes were selected to construct a prognostic model. The high-risk group showed significantly lower overall survival (OS), and this trend was observed in all subgroups: different age groups, genders, stages, and grades. Risk score was a risk factor independent of age, gender, stage, and grade. Moreover, the ICGC cohort validated the prognostic value of the model corresponding to the TCGA. In our cohort, qRT-PCR and immunohistochemistry showed that all three genes had higher expression levels in HCC samples than in normal controls. High expression levels of genes and high-risk scores showed significantly lower recurrence-free survival (RFS) and OS, especially in MVI-positive HCC samples. Therefore, the prognostic model constructed by three MVIRGs can reliably predict the RFS and OS of patients with HCC and is valuable for guiding clinical treatment selection and prognostic assessment of HCC.     

Heterologous expression of Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) enhanced chilling tolerance in transgenic eggplant (Solanum melongena L.)

Key message

Our study shows that the expression of AtCBF3 and AtCOR15A improved the chilling tolerance in transgenic eggplant.

Abstract

In an attempt to improve chilling tolerance of eggplant (Solanum melongena L) plants, Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) genes both driven by an Arabidopsis RESPONSIVE TO DESSICATION 29A promoter (AtRD29A) were transferred into the plants of eggplant cultivar Sanyueqie. Two independent homozygous transgenic lines were tested for their cold tolerance. The leaves of the transgenic plants in both lines withered much slower and slighter than the wild-type plants after exposure to cold stress treatment at 2 ± 1 °C. The gene expression of AtCBF3 and AtCOR15A was significantly increased as well as the proline content and the levels of catalase and peroxidase activities, while the relative electrical conductivity and the malondialdehyde content were remarkably decreased in the transgenic plants compared with the wild type at 4 ± 0.5 °C. The results showed that the expression of the exogenous AtCBF3 and AtCOR15A could promote the cold adaptation process to protect eggplant plants from chilling stress.     

Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles

The in vivo behavior of shell cross-linked knedel-like (SCK) nanoparticles is shown to be tunable via a straightforward and versatile process that advances SCKs as attractive nanoscale carriers in the field of nanomedicine. Tuning of the pharmacokinetics was accomplished by grafting varied numbers of methoxy-terminated poly(ethylene glycol) (mPEG) chains to the amphiphilic block copolymer precursors, together with chelators for the radioactive tracer and therapeutic agent (64)Cu, followed by self-assembly into block copolymer micelles and chemical cross-linking throughout the shell regions. (64)Cu-radiolabeling was then performed to evaluate the SCKs in vivo by means of biodistribution experiments and positron emission tomography (PET). It was found that the blood retention of PEGylated SCKs could be tuned, depending on the mPEG grafting density and the nanoparticle surface properties. A semiquantitative model of the density of mPEG surface coverage as a function of in vivo behavior was applied to enhance the understanding of this system.