Simple consensus procedures are effective and sufficient in secondary structure prediction

We have analyzed the performance of majority voting on minimal combination sets of three state-of-the-art secondary structure prediction methods in order to obtain a consensus prediction. Using three large benchmark sets from the EVA server, our results show a significant improvement in the average Q3 prediction accuracy of up to 1.5 percentage points by consensus formation. The application of an additional trivial filtering procedure for predicted secondary structure elements that are too short, does not significantly affect the prediction accuracy. Our analysis also provides valuable insight into the similarity of the results of the prediction methods that we combine as well as the higher confidence in consistently predicted secondary structure.     

Internalization and nuclear localization of interleukin 1 are not sufficient for function.

The human fibroblast interleukin 1 (IL-1) receptor is a glycosylated transmembrane protein with a cytoplasmic domain of 213 amino acids. We have constructed a series of deletion mutants of the cytoplasmic region of the IL 1 receptor and have used these mutants to examine its role in ligand binding, internalization, signal transduction, and nuclear localization of IL-1. Mutant receptors lacking most of the cytoplasmic domain are expressed at the cell surface and can bind, internalize, and localize IL-1 at the nucleus, but they do not allow IL-1-mediated induction of interleukin 2 and SV40 promoters. We have localized a critical region for signal transduction to a 50-amino acid segment of the cytoplasmic domain of the receptor. These studies demonstrate that IL-1 internalization and nuclear localization are not sufficient to trigger IL-1 activation of gene expression in T-cells.     

Within-compound associations are not sufficient to produce taste-mediated odor potentiation

Rats were used in two flavor-aversion experiments to determine if within-compound associations could be detected with a taste+odor compound that would not support taste-mediated odor potentation. In Experiment 1, following taste+odor compound conditioning, postconditioning taste extinction significantly weakened the odor aversion. In Experiment 2, following taste+odor compound conditioning, postconditioning taste inflation significantly strengthened the odor aversion. There was no evidence that taste potentiated the odor aversion in either Experiment 1 or 2. Thus, the results demonstrate that the presence of within-compound associations is not sufficient to produce taste-mediated odor potentiation. We offer a mediated conditioning explanation to account for the results of these two experiments.     

T cells are not sufficient for resistance to Pneumocystis carinii pneumonia in mice.

Severe combined immunodeficient (SCID) mice given spleen cells from immunocompetent donors resolve their pre-existing Pneumocystis carinii pneumonia (PCP). However, SCID mice given infusions of thymus cells or spleen cells depleted of cells positive for either immunoglobulin or immune response-associated antigen did not resolve their PCP. Immunofluorescence staining and mitogen responses of spleen cells from the recipient SCID mice confirmed that all groups of mice contained functional T cells but only the mice, reconstituted with nondepleted spleen cells, contained B cells. These results indicate, that T cells alone are not sufficient to resolve PCP in SCID mice and that B cells probably must also be present.     

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

The calcium concentration, [Ca]i, in single rat peritoneal mast cells was measured by means of the new Ca indicator dye fura-2. Upon stimulation with antigen or compound 48/80, [Ca]i rose for seconds to values greater than 5 microM. These Ca transients did not depend on the presence of extracellular Ca, and they sometimes occurred spontaneously, especially in the presence of exogenous phosphatidylserine. Calcium transients did not necessarily lead to degranulation. Degranulation usually occurred during periods of somewhat elevated [Ca] (0.5-1 microM) following transients but was sometimes observed at [Ca]i less than or equal to 250 nM. We found no evidence that an antigen-induced Ca influx is required for degranulation.     

MHC odours are not required or sufficient for recognition of individual scent owners

To provide information about specific depositors, scent marks need to encode a stable signal of individual ownership. The highly polymorphic major histocompatibility complex (MHC) influences scents and contributes to the recognition of close kin and avoidance of inbreeding when MHC haplotypes are shared. MHC diversity between individuals has also been proposed as a primary source of scents used in individual recognition. We tested this in the context of scent owner recognition among male mice, which scent mark their territories and countermark scents from other males. We examined responses towards urine scent according to the scent owner's genetic difference to the territory owner (MHC, genetic background, both and neither) or genetic match to a familiar neighbour. While urine of a different genetic background from the subject always stimulated greater scent marking than own, regardless of familiarity, MHC-associated odours were neither necessary nor sufficient for scent owner recognition and failed to stimulate countermarking. Urine of a different MHC type to the subject stimulated increased investigation only when this matched both the MHC and genetic background of a familiar neighbour. We propose an associative model of scent owner recognition in which volatile scent profiles, contributed by both fixed genetic and varying non-genetic factors, are learnt in association with a stable involatile ownership signal provided by other highly polymorphic urine components.     

Rare codons are not sufficient to destabilize a reporter gene transcript in tobacco

In plants, as in other eukaryotes, most synonymous codons of the genetic code are not used with equal frequency, but instead some codons are preferred, whereas others are rare. Circumstantial evidence led to the suggestion that rare codons have a negative influence on mRNA stability. To address this question experimentally, rare codons encoded by a Bacillus thuringiensis (B.t.) toxin gene (cryIA(c)) or a synthetic sequence were introduced into a phytohemagglutinin (PHA) reporter gene. In neither case was the mRNA stability appreciably diminished in stably transformed tobacco cell cultures nor was the accumulation of mRNA in transgenic plants affected. Thus rare codons do not appear to be sufficient to cause rapid degradation of the PHA mRNA and potentially other mRNAs in plants.     

Cutting edge: TLR ligands are not sufficient to break cross-tolerance to self-antigens

Cross-presentation of peripheral self-Ags by dendritic cells (DC) can induce deletion of autoreactive CTL by a mechanism termed cross-tolerance. Activation of DC by microbial TLR ligands is thought to result in adaptive immunity. However, activation of tolerogenic DC may cause autoimmunity by stimulating instead of deleting autoreactive CTL. To investigate this scenario, we have monitored the response of autoreactive CTL in specific for the transgenic self Ag, OVA, expressed in pancreatic islets of RIP-mOVA mice injected with ligands of TLR2, 3, 4, and 9. This somewhat enhanced proliferation and cytokine production, and moderately reduced the CTL number able to induce autoimmunity. Nevertheless, physiological CTL numbers were deleted before disease ensued, unless specific CD4 T cell help was provided. In conclusion, DC activation by TLR ligands was insufficient to break peripheral cross-tolerance in the absence of specific CD4 T cell help, and triggered autoimmunity by stimulating the early effector phase of autoreactive CTL only when their precursor frequency was extremely high.     

Nucleotides that are essential but not conserved; a sufficient L-tryptophan site in RNA

Conservation is often used to define essential sequences within RNA sites. However, conservation finds only invariant sequence elements that are necessary for function, rather than finding a set of sequence elements sufficient for function. Biochemical studies in several systems—including the hammerhead ribozyme and the purine riboswitch—find additional elements, such as loop–loop interactions, required for function yet not phylogenetically conserved. Here we define a critical test of sufficiency: We embed a minimal, apparently sufficient motif for binding the amino acid tryptophan in a random-sequence background and ask whether we obtain functional molecules. After a negative result, we use a combination of three-dimensional structural modeling, selection, designed mutations, high-throughput sequencing, and bioinformatics to explore functional insufficiency. This reveals an essential unpaired G in a diverse structural context, varied sequence, and flexible distance from the invariant internal loop binding site identified previously. Addition of the new element yields a sufficient binding site by the insertion criterion, binding tryptophan in 22 out of 23 tries. Random insertion testing for site sufficiency seems likely to be broadly revealing.     

Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white‐nose syndrome

White‐nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans (Pd) that affects bats during hibernation. Although millions of bats have died from WNS in North America, mass mortality has not been observed among European bats infected by the fungus, leading to the suggestion that bats in Europe are immune. We tested the hypothesis that an antibody‐mediated immune response can provide protection against WNS by quantifying antibodies reactive to Pd in blood samples from seven species of free‐ranging bats in North America and two free‐ranging species in Europe. We also quantified antibodies in blood samples from little brown myotis (Myotis lucifugus) that were part of a captive colony that we injected with live Pd spores mixed with adjuvant, as well as individuals surviving a captive Pd infection trial. Seroprevalence of antibodies against Pd, as well as antibody titers, was greater among little brown myotis than among four other species of cave‐hibernating bats in North America, including species with markedly lower WNS mortality rates. Among little brown myotis, the greatest titers occurred in populations occupying regions with longer histories of WNS, where bats lacked secondary symptoms of WNS. We detected antibodies cross‐reactive with Pd among little brown myotis naïve to the fungus. We observed high titers among captive little brown myotis injected with Pd. We did not detect antibodies against Pd in Pd‐infected European bats during winter, and titers during the active season were lower than among little brown myotis. These results show that antibody‐mediated immunity cannot explain survival of European bats infected with Pd and that little brown myotis respond differently to Pd than species with higher WNS survival rates. Although it appears that some species of bats in North America may be developing resistance to WNS, an antibody‐mediated immune response does not provide an explanation for these remnant populations.     

Soluble oligomers are sufficient for transmission of a yeast prion but do not confer phenotype

Amyloidogenic proteins aggregate through a self-templating mechanism that likely involves oligomeric or prefibrillar intermediates. For disease-associated amyloidogenic proteins, such intermediates have been suggested to be the primary cause of cellular toxicity. However, isolation and characterization of these oligomeric intermediates has proven difficult, sparking controversy over their biological relevance in disease pathology. Here, we describe an oligomeric species of a yeast prion protein in cells that is sufficient for prion transmission and infectivity. These oligomers differ from the classic prion aggregates in that they are soluble and less resistant to SDS. We found that large, SDS-resistant aggregates were required for the prion phenotype but that soluble, more SDS-sensitive oligomers contained all the information necessary to transmit the prion conformation. Thus, we identified distinct functional requirements of two types of prion species for this endogenous epigenetic element. Furthermore, the nontoxic, self-replicating amyloid conformers of yeast prion proteins have again provided valuable insight into the mechanisms of amyloid formation and propagation in cells.     

Cell-adhesion and morphological changes are not sufficient to support anchorage-dependent cell growth via non-integrin-mediated attachment

Cell-adhesion and spread are important for cell survival. Although extensive studies have suggested several potential mechanisms of action, it is not yet clear how important cell-morphological change per se contributes to the cell-surviving signal. We employed a non-integrin-mediated cell-adhesion system to explore this question. BHK-Japanese encephalitis virus (JEV) cells (BHK21 cells that are persistently infected with JEV) express a large amount of JEV-envelope protein (JEV E) on their surfaces, and can attach and form pseudopodia on the anti-JEV E antibody-coated substrates. However, cells that adhered on the antibody substrate underwent a caspase-3-mediated apoptosis together with a down-regulation of mitogen-activated protein kinase activity within 20 h after adhesion, which indicates that viral-protein-mediated cell-adhesion and cell-spread are not sufficient for supporting cell survival. This provides a different perspective for the study of the relationships between the cell-morphological change and the cell-survival signal.     

Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury

Background

Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury.

Methods and Findings

Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH⁺ and Iba-1⁺ cells acutely died, and as the penumbra the area surrounding the core where Iba-1⁺ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d.

Conclusions

Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic.