Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.     

Four alkaloids, lucidine B, oxolucidine A, lucidine A, and lucidulinone from Lycopodium lucidulum

The structures of four alkaloids extracted from Lycopodium lucidulum (Lycopodiaceae) were established by X-ray and 2D NMR spectroscopic analyses. The dihydro-derivative of oxolucidine A, which was obtained by NaBH4 reduction of oxolucidine A, was treated with p-bromobenzoyl chloride to afford crystals, whose X-ray crystallographic analysis established the stereostructure, including the absolute configuration. The 2D NMR spectra of tetrahydrodeoxylucidine B were fully analyzed to establish the full structure of lucidine B, and the hitherto unknown stereochemistry at the C-14 position was established as beta-H. The structure of a new alkaloid, lucidulinone, was determined by spectroscopic analysis to be luciduline lactam.     

Kin selection does not explain male aggregation at leks of 4 manakin species

In lek-mating systems, males aggregate at display arenas andfemales visit solely for the purpose of mating. This breedingsystem is characterized by high variance in male mating successwith one male often receiving most copulations. High reproductiveskew among males has led to question why males join leks whentheir chances of reproductive success are so low. Kin selectionhas been invoked as a mechanism to explain the evolution oflekking behavior, whereby nonreproducing but genetically relatedmales gain indirect inclusive-fitness benefits. Evidence forkin selection among lek-mating birds is, however, mixed. Here,we show that kin selection is unlikely to be an important explanationfor evolution of lekking behavior in manakins (Aves: Pipridae).We found that for 4 species chosen from several major cladeswithin Pipridae, males within leks were not significantly morerelated than expected from random assortment of males in thepopulation. This means that nonreproducing males do not gainindirect inclusive-fitness benefits by joining leks. This resultsuggests alternative mechanisms must be invoked to explain theevolution of lek-mating systems in manakins.     

A study of glycoproteins in human serum and plasma reference standards (HUPO) using multilectin affinity chromatography coupled with RPLC-MS/MS

The glycoproteome is a major subproteome present in human plasma. In this study, we isolated and characterized approximately 150 glycoproteins from the human plasma and serum samples provided by HUPO using a multilectin affinity column. The corresponding tryptic digest was separated by RP-HPLC coupled to an IT mass spectrometer (3-D LCQ). Also in this study, a new system, namely an Ettan MDLC system coupled to a linear ITLTQ, was compared with the previous LCQ platform and gave a greater number of protein identifications, as well as better quality. When we compared the composition of the glycoproteomes for the plasma and serum samples there was a close correlation between the samples, except for the absence of fibrinogen from the identified-protein list in the latter sample, which was presumably as a result of the clotting process. In addition, the analysis of the samples from three ethnic specimens, Caucasian American, Asian American, and African American, were very similar but showed a higher angiotensinogen plasma level and a lower histidine-rich glycoprotein level in Caucasian American samples, and a lower vitronectin level in African American blood samples.     

Inhibition of mechanistic target of rapamycin promotes dendritic cell activation and enhances therapeutic autologous vaccination in mice

Dendritic cells (DCs) are potent inducers of T cell immunity, and autologous DC vaccination holds promise for the treatment of cancers and chronic infectious diseases. In practice, however, therapeutic vaccines of this type have had mixed success. In this article, we show that brief exposure to inhibitors of mechanistic target of rapamycin (mTOR) in DCs during the period that they are responding to TLR agonists makes them particularly potent activators of naive CD8(+) T cells and able to enhance control of B16 melanoma in a therapeutic autologous vaccination model in the mouse. The improved performance of DCs in which mTOR has been inhibited is correlated with an extended life span after activation and prolonged, increased expression of costimulatory molecules. Therapeutic autologous vaccination with DCs treated with TLR agonists plus the mTOR inhibitor rapamycin results in improved generation of Ag-specific CD8(+) T cells in vivo and improved antitumor immunity compared with that observed with DCs treated with TLR agonists alone. These findings define mTOR as a molecular target for augmenting DC survival and activation, and document a novel pharmacologic approach for enhancing the efficacy of therapeutic autologous DC vaccination.     

Short- and long-term effects of (+)-methamphetamine and (+/-)-3,4-methylenedioxymethamphetamine on monoamine and corticosterone levels in the neonatal rat following multiple days of treatment

Rats treated with (±)-3,4-methylenedioxymethamphetamine (MDMA) or (+)-methamphetamine (MA) neonatally exhibit long-lasting learning impairments (i.e., after treatment on postnatal days (P)11–15 or P11–20). Although both drugs are substituted amphetamines, they each produce a unique profile of cognitive deficits (i.e., spatial vs. path integration learning and severity of deficits) which may be the result of differential early neurochemical changes. We previously showed that MA and MDMA increase corticosterone (CORT) and MDMA reduces levels of serotonin (5-HT) 24 h after treatment on P11, however, learning deficits are seen after 5 or 10 days of drug treatment, not just 1 day. Accordingly, in the present experiment, rats were treated with MA or MDMA starting on P11 for 5 or 10 days (P11–15 or P11–20) and tissues collected on P16, P21, or P30. Five-day MA administration dramatically increased CORT on P16, whereas MDMA did not. Both drugs decreased hippocampal 5-HT on P16 and P21, although MDMA produced larger reductions. Ten-day treatment with either drug increased dopamine utilization in the neostriatum on P21, whereas 5-day treatment had no effect. No CORT or brain 5-HT or dopamine changes were found with either drug on P30. Although the monoamine changes are transient, they may alter developing neural circuits sufficiently to permanently disrupt later learning and memory abilities.