A Bethlem myopathy Gly to Glu mutation in the von Willebrand factor A domain N2 of the collagen alpha3(VI) chain interferes with protein folding.

A single G1679E mutation in the amino-terminal globular domain N2 of the alpha3 chain of type VI collagen was found in a large family affected with Bethlem myopathy. Recombinant production of N2 ( approximately 200 residues) in transfected mammalian cells has now been used to examine the possibility that the mutation interfered with protein folding. The wild-type form and a G1679A mutant were produced at high levels and shown to fold into a stable globular structure. Only a small amount of secretion was observed for mutants G1679E and G1679Q, which apparently were efficiently degraded within the cells. Homology modeling onto the related von Willebrand factor A1 structure indicated that substitution of G1679 by the bulky E or Q cannot be accommodated without considerable changes in the folding pattern. This suggests protein misfolding as a molecular basis for this particular mutation in Bethlem myopathy, in agreement with radioimmunoassay data showing reduced levels of domain N2 in cultured fibroblasts from two patients.     

Chimeric antigen receptor engineered innate immune cells in cancer immunotherapy

Introducing chimeric antigen receptor into immune cells against malignancies has contributed to a revolutionary innovation in cancer immunotherapy. As an important type of adaptive immune cells, T cells first caught researchers' attention and became great success in chimeric antigen receptor-based immunotherapy. However, engineered T cells seem to hit their bottleneck when resistance of cancerous cells, less encouraging responses in solid tumors and unwanted toxicities to the host remain to be solved.Meanwhile, innate immune cells get to join the race. Representatives such as natural killer cells, natural killer T cells, γδT cells and macrophages also prove to be well redirected with chimeric antigen receptors. Compared to chimeric antigen receptor engineered T cells, these engineered innate immune cells may possess multiple targeting and killing mechanisms, have the potential to crack the barrier of solid tumors and have less side effects in the host. Besides, possible universal access to cell resources and improvements in expansion and transduction techniques make these cells promising candidates with huge potential in translational medicine. Therefore, innate immune cells claim a brand-new dimension and are likely to supplement T cells greatly in the field of chimeric antigen receptor-based immunotherapy.     

Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.     

The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA

Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.     

Specificity and inhibition studies of human renal dipeptidase

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)