Expression of the gene encoding the adenovirus DNA terminal protein precursor in productively infected and transformed cells.

The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.     

Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

Arm anomalies: cases of supernumerary development and bilateral agenesis of arm pairs in Octopoda (Mollusca,Cephalopoda)

Summary The first case of true hexapody among the Octopoda, resulting from bilateral agenesis of one arm pair, is described for a malePteroctopus tetracirrhus. A case of decapody, with uniform development of all arms, is also reported for the first time for a maleOctopus briareus. Both conditions apparently result from developmental anomalies of the embryonic arm anlagen. A survey of other anomalous conditions relating to arm development and regeneration within the Cephalopoda is provided. A possible relationship of polyfurcation of arm tips in the Octopoda with regenerative processes in amphibian limbs leading to similar conditions is suggested.     

A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.     

L-Selenocysteine induced HepG-2 cells apoptosis through reactive oxygen species-mediated signaling pathway

Molecular Biology Reports - Currently, Liver cancer is the fifth most common tumor and the second most important reason for cancer-related death in the world. However, there are still many...