Distinct and Cooperative Activities of HESO1 and URT1 Nucleotidyl Transferases in MicroRNA Turnover in Arabidopsis

3’ uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2’-O-methylation on the 3’ terminal ribose protects micro(mi)RNAs from 3’ truncation and 3’ uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3’ tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3’ end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3’ tailing could affect the activities of miRNAs in addition to leading to miRNA degradation.     

Genetic variation and population structure in native Americans

We examined genetic diversity and population structure in the American landmass using 678 autosomal microsatellite markers genotyped in 422 individuals representing 24 Native American populations sampled from North, Central, and South America. These data were analyzed jointly with similar data available in 54 other indigenous populations worldwide, including an additional five Native American groups. The Native American populations have lower genetic diversity and greater differentiation than populations from other continental regions. We observe gradients both of decreasing genetic diversity as a function of geographic distance from the Bering Strait and of decreasing genetic similarity to Siberians--signals of the southward dispersal of human populations from the northwestern tip of the Americas. We also observe evidence of: (1) a higher level of diversity and lower level of population structure in western South America compared to eastern South America, (2) a relative lack of differentiation between Mesoamerican and Andean populations, (3) a scenario in which coastal routes were easier for migrating peoples to traverse in comparison with inland routes, and (4) a partial agreement on a local scale between genetic similarity and the linguistic classification of populations. These findings offer new insights into the process of population dispersal and differentiation during the peopling of the Americas.     

Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this study, we have developed chemoenzymatic synthesis of extended MUC1 TR glycopeptides with cancer-associated O-glycosylation using a panel of recombinant human glycosyltransferases. MUC1 glycopeptides with different densities of Tn and STn glycoforms conjugated to KLH were used as immunogens to evaluate an optimal vaccine design. Glycopeptides with complete O-glycan occupancy (five sites per repeat) elicited the strongest antibody response reacting with MUC1 expressed in breast cancer cell lines in both Balb/c and MUC1.Tg mice. The elicited humoral immune response showed remarkable specificity for cancer cells suggesting that the glycopeptide design holds promise as a cancer vaccine. The elicited immune responses were directed to combined glycopeptide epitopes, and both peptide sequence and carbohydrate structures were important for the antigen. A MAb (5E5) with similar specificity as the elicited immune response was generated and shown to have the same remarkable cancer specificity. This antibody may hold promise in diagnostic and immunopreventive measures.     

Evaluation of transgenic tobacco plants expressing a bacterial Co-Ni transporter for acquisition of cobalt

Phytoremediation is a viable strategy for management of toxic wastes in a large area/volume with low concentrations of toxic elemental pollutants. With increased industrial use of cobalt and its alloys, it has become a major metal contaminant in soils and water bodies surrounding these industries and mining sites with adverse effects on the biota. A bacterial Co-Ni permease was cloned from Rhodopseudomonas palustris and introduced into Nicotiana tabacum to explore its potential for phytoremediation and was found to be specific for cobalt and nickel. The transgenic plants accumulated more cobalt and nickel as compared to control, whereas no significant difference in accumulation of other divalent ions was observed. The transgenic plants were evaluated for cobalt content and showed increased acquisition of cobalt (up to 5 times) as compared to control. The plants were also assessed for accumulation of nickel and found to accumulate up to 2 times more nickel than control. At the same initial concentration of cobalt and nickel, transgenic plant preferentially accumulated cobalt as compared to nickel. The present study is perhaps the first attempt to develop transgenic plants expressing heterologous Co transporter with an improved capacity to uptake cobalt.     

Proteinase-activated receptors (PARs): differential signalling by kallikrein-related peptidases KLK8 and KLK14

We compared signalling pathways such as calcium transients, MAPK activation, β-arrestin interactions and receptor internalization triggered by kallikrein-related peptidases (KLKs) 8 and 14 in human and rat proteinase-activated receptor (PAR)2-expressing human embryonic kidney (HEK) and Kirsten transformed rat kidney (KNRK) cells. Further, we analysed processing by KLK8 vs. KLK14 of synthetic human and rat PAR2-derived sequences representing the cleavage-activation domain of PAR2. Our data show that like KLK14, KLK8 can unmask a PAR2 receptor-activating sequence from a peptide precursor. However, whilst KLK8, like KLK14, can signal in rat-PAR2-expressing KNRK cells, this enzyme cannot signal via human PAR2 in HEK or KNRK cells, but rather, disarms HEK PAR1. Thus, KLK8 and KLK14 can signal differentially via the PARs to affect tissue function.     

Highly-restricted, cell-specific expression of the simian CMV-IE promoter in transgenic zebrafish with age and after heat shock

Promoters with high levels of ubiquitous expression are of significant utility in the production of transgenic animals and cell lines. One such promoter is derived from the human cytomegalovirus immediate early (CMV-IE) gene. We sought to ascertain if the simian CMV-IE promoter (sCMV), used extensively in non-mammalian vertebrate research, also directs intense, widespread expression when stably introduced into zebrafish. Analysis of sCMV-driven expression revealed a temporal and spatial pattern not predicted by studies using the hCMV promoter in other transgenic animals or by observations of early F0 embryos expressing injected sCMV-reporter plasmids. Unexpectedly, in transgenic fish produced by both integration of linearized plasmid or Tol2-mediated transgenesis, sCMV promoter expression was generally observed in a small population of cells in telencephalon and spinal cord between days 2 and 7, and was thereafter confined to discrete regions of CNS that included the olfactory bulb, retina, cerebellum, spinal cord, and lateral line. In skeletal muscle, intense transgene expression was not observed until well into adulthood (>2-3 months post-fertilization). One final unexpected characteristic of the sCMV promoter in stable transgenic fish was tissue-specific responsiveness of the promoter to heat shock at both embryonic and adult stages. These data suggest that, in the context of stable transgenesis, the simian CMV-IE gene promoter responds differently to intracellular regulatory forces than other characterized CMV promoters.     

Bioactive Peptides from Marine Ascidians and Future Drug Development–A Review

Marine ascidians are considered as one of the richest sources of bioactive compounds. The extraction and utilization of marine peptides have been attracted much attention owing to their potential health benefits. Most of the bioactive compounds from marine ascidians are already in different phases of the clinical and preclinical pipeline. They can be used in different functional and nutraceutical values due to their antineoplastic, antihypertensive, antioxidant, and antimicrobial properties. The screening in vivo and in vitro bioassays are coupled to the purification process for the exploration of its biological interest which is of great value. The growing significance to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs and other drugs are in clinical/pre-clinical trials. The present review highlights the recent research progress in marine ascidians’ peptides and its prospects for the future pharmaceutical development.