Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the deltaSH virus was only marginally less efficient than that of wild-type HMPV, whereas the deltaG and deltaM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The deltaG and deltaM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.     

Seed weight increases with altitude in the Swiss Alps between related species but not among populations of individual species

Seed weight is a crucial plant life history trait, determining establishment success and dispersal ability. Especially in stressful environments, larger seeds may be selected at the expense of seed number, because larger seeds have a better chance of giving rise to an established offspring. We tested the hypotheses that between related species-pairs and among populations of single species a similar trend for increasing seed weight with increasing altitude should be present. Firstly, we measured seed weights from 29 species-pairs, with one species occurring in lowland areas and a congeneric species from high altitudes. Seeds of the alpine species were 28±8% larger than seeds from lowland species (P<0.01). Compared to the related lowland species, 55% of the alpine species had heavier seeds, 3% (one species) had lighter, and 41% had seeds of approximately equal weight. Secondly, we compared seed weights among populations of four species from different habitats and with different life histories. Seeds from between 11 and 34 populations per species were sampled along altitudinal gradients of 800–1,500 m (ca. 800 m in Scabiosa lucida, ca. 1,000 m in Saxifraga oppositifolia, ca. 1,000 m in Epilobium fleischeri, and ca. 1,500 m in Carex flacca). In all the four species, we found no indication for heavier seeds at higher altitudes. Our results indicate a selection pressure for species with heavier seeds at higher altitude, but the trend does not seem to operate across all cases. Phylogenetic constraints may limit the correlation among altitude and seed weight, operating particularly against selection for larger seed size, the closer populations and species are related to each other.     

Distinguishing between metabolically active and inactive roots by combined staining with 2,3,5-triphenyltetrazolium chloride and image colour analysis

The objective of the present work was to develop a method to distinguish between metabolically inactive and active parts of plant roots. White clover (Trifolium repens L.) roots were stained with 2,3,5-triphenyltetrazolium chloride (TTC) followed by root colour classification with an interactive scanner-based image analysis programme (WinRHIZO). Roots inactivated by boiling were unstained and pale brown, whereas fresh samples with predominantly metabolically active roots turned dark red, red or pale red after staining. A small amount of very young, presumable active roots (0.8% of total active root length) failed to stain red with TTC. The colour analysis of inactive and active roots was based on four colour classes for boiled roots and seven classes for fresh roots, respectively, as defined upon visual examination of images. Pixel colours falling outside the defined classes were allocated to the nearest defined class – an option that increased objectivity and stability and reduced the required number of colour classes. For the fresh white clover roots, 75–86% of the total root length was determined as active, while 3–7% of the boiled roots fell into the same category. The percentage of total root length measured by WinRHIZO that was identified as metabolically active was linearly correlated with the percentage of fresh roots in mixtures of fresh and boiled roots (R²=0.99). Colour classes chosen à priori from one experiment could be used to distinguish fairly satisfactorily between active and inactive roots of another white clover cultivar grown under other conditions, but failed to classify activity in ryegrass (Lolium multiflorum Lam.) root samples. In the latter case, colour classes needed to be re-defined in order to produce reliable data. Our work shows that WinRHIZOs colour identification sub-module provides a new promising tool to classify root activity as identified after staining with TTC, but colour classes must be carefully evaluated on every new occasion.     

Aberrant heteroplasmic transmission of mtDNA in cloned pigs arising from double nuclear transfer

Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.     

Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.     

fMRI Hippocampal Activity During a VirtualRadial Arm Maze

Numerous studies have shown that the hippocampus is critical for spatial memory. Within nonhuman research, a task often used to assess spatial memory is the radial arm maze. Because of the spatial nature of this task, this maze is often used to assess the function of the hippocampus. Our goal was to extrapolate this task to humans and examine whether healthy undergraduates utilize their hippocampus while performing a virtual reality version of the radial arm maze task. Thirteen undergraduates performed a virtual radial arm maze during functional magnetic resonance imaging. The brain maps of activity reveal bilateral hippocampal BOLD signal changes during the performance of this task. However, paradoxically, this BOLD signal change decreases during the spatial memory component of the task. Additionally, we note frontal cortex activity reflective of working memory circuits. These data reveal that, as predicted by the rodent literature, the hippocampus is involved in performing the virtual radial arm maze in humans. Hence, this virtual reality version may be used to assess the integrity of hippocampus so as to predict risk or severity in a variety of psychiatric disorders.     

Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms

The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.     

Arginine methylation an emerging regulator of protein function

Arginine methylation is now coming out of the shadows of protein phosphorylation and entering the mainstream, largely due to the identification of the family of enzymes that lay down this modification. In addition, modification-specific antibodies and proteomic approaches have facilitated the identification of an array of substrates for the protein arginine methyltransferases. This review describes recent insights into the molecular processes regulated by arginine methylation in normal and diseased cells.