Long‐term response of temperate canopy trees to removal of browsing from an invasive arboreal herbivore in New Zealand

Defoliation of forest tree canopies by herbivores and other agents, leading to tree mortality and reduced productivity, threatens the ecological stability of forests globally. This study shows that long‐term control of a mammalian arboreal folivore (brushtail possums; Trichosurus vulpecula Phalangeridae) reduces crown dieback and increases foliage cover in browsing‐damaged canopy trees. We monitored indices of possum density, possum browsing, tree foliage cover and crown dieback for 20 years following initiation of possum control in 1994 that repeatedly reduced possum densities to near zero every 5–6 years and kept the population below 35% of pre‐control levels over the entire period. Observable possum browsing was recorded on 20–49% of individuals of three palatable tree species at the time of first control. Those percentages fell to zero after control and never exceeded 2–10% for individual species over the next 19 years. We recorded significant increases in foliage cover attributable to recovery from defoliation by possums for all three species during the first 10 years. Large increases in foliage cover occurred on individuals that were heavily browsed in 1994 (mean increases: 36–89%), but mean population increases were modest (3–19%) because only 10–19% of trees were initially heavily browsed. Twenty‐year mortality rates were similar for plants with, or without, initial possum browsing, indicating no residual impact of pre‐control browsing on tree mortality. Times for full recovery of crown foliage cover varied from 10 years for the youngest trees and faster growing species to more than 20 years for mature individuals of the slowest growing species.     

Measurement of imino <Superscript>1</Superscript>H–<Superscript>1</Superscript>H residual dipolar couplings in RNA

Imino ¹H–¹⁵N residual dipolar couplings (RDCs) provide additional structural information that complements standard ¹H–¹H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino ¹H–¹H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA^Val using a BEST-Jcomp-HMQC2 experiment. ¹H–¹H RDCs are observed between the imino protons in G–U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino ¹H–¹H RDCs complement standard ¹H–¹⁵N RDCs because the ¹H–¹H vectors generally point along the helical axis, roughly perpendicular to ¹H–¹⁵N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino ¹H–¹H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of early changes in autoimmune T cell phenotype and function following intravenous administration of type II collagen in a TCR-transgenic model.

To study the phenotypic and functional changes in naive type II collagen (CII)-specific autoimmune T cells following a tolerogenic signal, a TCR-transgenic (Tg) mouse model of collagen-induced arthritis was developed. These Tg mice express an I-A(q)-restricted CII (260-267)-specific TCR that confers severe accelerated autoimmune arthritis following immunization with CII. Despite the fact that >90% of the alphabeta T cells express the Tg, these mice can be rendered completely tolerant to the induction of arthritis by i.v. administration of 200 microg of CII. As early as 24 h after CII administration, CII-specific T cells demonstrated a decreased ability to proliferate in response to the CII immunodominant peptide and phenotypically altered the expression of L-selectin to CD62L(low) and of phagocytic glycoprotein-1 to CD44(high), expression levels consistent with the phenotype of memory T cells. In addition, they up-regulated the expression of the activation markers CD71 and CD69. Functionally, following tolerogenic stimulation, the CII-specific T cells produced similar levels of IL-2 in comparison to controls when challenged with CII peptide, however, by 48 h after exposure to tolerogen, IL-2 production dropped and was replaced by high levels of IL-10 and IL-4. Based on their production of Th2 cytokines, these data suggest that T regulatory cells expressing activation and memory markers are induced by the tolerogen and may exert their influence via cytokines to protect the animals from the induction of arthritis.     

Blockade of virus infection by human CD4+ T cells via a cytokine relay network

CD4(+) T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8(+) T cell functions, we sought to determine whether human CD4(+) T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-gamma and TNF-alpha, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-gamma and TNF-alpha acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-gamma and TNF-alpha also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-gamma and TNF-alpha secreted by human Th1 cells acted indirectly through the IFN-alpha/beta receptor. These results highlight the importance of CD4(+) T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.     

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.     

A screen of random sequences for those that alter the trafficking of the influenza virus hemagglutinin in vivo

In order to determine if the sequence patterns known to specify internalization represent the majority of possible internalization signals, we identified random sequences capable of causing a reporter protein to be internalized at least several-fold faster than the rate of non-selective internalization of membrane by clathrin-coated pits. A library of influenza hemagglutinin (HA) proteins, bearing short random sequences in place of the wild-type cytoplasmic domain, was prepared in recombinant SV40 virus. The library was expressed and screened for HAs that could internalize anti-HA antibody from the medium. The cytoplasmic sequences of the selected proteins were determined. From a small sample of sequences, we detected several that did not resemble those previously identified. The known internalization signals must represent only a subset of the sequences that can serve as internalization signals.