Metabolically Active Synaptosomes Can Be Prepared from Frozen Rat and Human Brain

Abstract: Nerve ending particles (synaptosomes) were prepared from pieces of rat and human brain and from brain homogenate that had been frozen and thawed under a variety of conditions. Their purity, as judged by electron microscopy, and performance in terms of a number of metabolic and functional parameters [accumulation of tissue potassium, respiration, release of transmitter amino acids, and the responses on these indices to depolarisation by veratrine (VX)] were compared with those of fresh tissue-derived synaptosomes. It was found that rapid freezing and/or slow thawing severely impaired the subsequent performance of incubated synaptosomes. In contrast, synaptosomes from tissue frozen slowly and thawed rapidly showed relatively good retention of morphology and metabolic performance. It was better to use whole (1-5 g) pieces of tissue than tissue homogenate: the synaptosome fraction from frozen tissue pieces contained 80% of the proportion of identified synaptosomes found in the fresh tissue synaptosome fraction, its respiratory rate was 65%, and its tissue potassium content 70% of that of fresh controls. Moreover, it responded to VX or potassium stimulation by showing increased respiratory rate, decreased tissue potassium, and increased release of neurotransmitter amino acids, to an extent that was comparable to that of fresh tissue fractions. Thus, preparations from frozen rat and human brain were shown to be metabolically and functionally active, and can be used for a variety of neurotransmitter-related studies.     

Toxicity of Quinacrine Can Be Reduced By Co-Administration of P-Glycoprotein Inhibitor in Sporadic Creutzfeldt-Jakob Disease

1. Recent publication has suggested that quinacrine may be a candidate for treatment of Creutzfeldt-Jakob disease (CJD). But serious toxicity of quinacrine to liver and hematological system has been reported.2. We disclosed the permeability of quinacrine can be enhanced by presence of p-glycoprotein inhibitor at blood–brain barrier in vitro. Therefore, we tried the protocol of combination of quinacrine and p-glycoprotein inhibitor, verapamil for patients with CJD.3. When compared clinical effects by quinacrine and the combination therapy, improvement of clinical findings was observed at the same level without any adverse effects. Low-dose quinacrine with verapamil can be used as safe treatment of CJD.     

Cellular Microvesicle Pathways Can Be Targeted to Transfer Genetic Information between Non-Immune Cells

Eukaryotic cell communication is based on protein signaling cascades that require direct cell-cell apposition, or receptor engagement by secreted molecules. The transmission of genetic information is thought to be uncommon, apart from recent reports of exosomal RNA transfer in immune and glioblastoma cells. We wished to examine if existing microvesicle pathways could be directly targeted for the horizontal transfer of RNA genomes in less specialized cell types. Using replication-deficient retrovirus vector, studies herein confirm that a range of cells routinely sequester a small population of these RNA genomes in a non-canonical compartment, refractory to antibody neutralization and unaffected by specific pharmacological inhibition of pathways involved in conventional viral trafficking. Our experiments further reveal the cytoplasmic colocalization of vector genomes with tetraspanin proteins as well as the PI-3-kinase sensitive trafficking and subsequent transmission to 2° targets. Collectively, our results indicate a scalable process whereby cells route vector genomes to multivesicular bodies (MVB) for cytoplasmic trafficking and exosomal release. Our findings imply that cells can serve to deliver recombinant payload, targeted for the stable genetic modification of 2° target cells.     

Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity

Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.Few of the hundreds of known neutralizing anti-HIV monoclonal antibodies (MAbs) display broad cross-reactive activities (4). Of those derived from clade B-infected patients, b12 binds to the gp120 subunit of the HIV envelope protein (Env), to an epitope that overlaps the CD4 binding site, and neutralizes approximately 50% of virus isolates tested, including non-clade B viruses (27). 2G12 binds to N-linked carbohydrates on gp120 (32, 34) and neutralizes 41% of isolates tested, although not clade C or E isolates. 447-52D also binds to the gp120 subunit, to an epitope within the V3 loop, and potently neutralizes up to 45% of clade B isolates but rarely non-clade B isolates. 4E10 and 2F5 recognize adjacent epitopes located at the membrane-proximal external region (MPER) of the gp41 Env subunit (9, 22, 24, 28, 42). Two neutralizing antibodies (NAbs) isolated from a clade A-infected patient (PG9 and PG16) show broad and potent neutralizing activity by recognizing epitopes consisting of conserved regions of the V2 and V3 loops of gp120, preferentially on native trimers (40).4E10 is capable of neutralizing all isolates tested at some level (4), although there is evidence for the existence of rare viruses that are resistant to 4E10 neutralization (30). The exact structure of the epitope recognized by 4E10 within the trimeric, functional HIV Env is unknown, but structural studies have shown that an isolated peptide spanning the epitope adopts a helical conformation, a short 3₁₀ segment followed by a 4₁₃ (or true α-helical) segment, with an extended structure at the N terminus when bound to 4E10 (9). It has also been reported that 4E10 interacts with a variety of lipids and membrane components, particularly the phospholipid cardiolipin (15), suggesting that difficulties in eliciting 4E10-like broadly neutralizing antibodies by immunization and the apparent rarity of 4E10-like antibody responses in HIV-1-infected subjects (19, 33) are linked to this polyspecificity to autoantigens, contributing to their elimination through tolerance mechanisms. However, subsequent studies have shown that the measurable, but quite weak, affinity of 4E10 for certain lipids is comparable to that of some antiphospholipid antibodies elicited during many infections, suggesting that 4E10 is not remarkably autoreactive (35). Therefore, it is still unclear whether lipid binding properties are linked to the rarity of 4E10-like specificities. It has also been proposed that the neutralizing activity of 4E10 may partly depend on lipid binding, either through interactions with viral membrane lipids that disturb the membrane-bound structure of the MPER on the trimeric, virion-associated Env spike (39) or through an encounter model. In the latter, initial interactions with membrane components align 4E10 with its protein epitope or allow 4E10 to gain proximity to its epitope (1), perhaps partially alleviating steric occlusion effects (for example, see reference 17). We sought to determine whether specific interactions exist between 4E10 and membrane lipid components and whether such interactions meaningfully contribute to neutralization by any mechanism.     

Myxoma Virus Oncolytic Efficiency Can Be Enhanced Through Chemical or Genetic Disruption of the Actin Cytoskeleton

Myxoma virus (MYXV) is one of many animal viruses that exhibit oncolytic properties in transformed human cells. Compared to orthopoxviruses like vaccinia (VACV), MYXV spreads inefficiently, which could compromise its use in treating tumors and their associated metastases. The VACV F11 protein promotes virus exit and rapid spread by inhibiting Rho signalling, which results in a disruption of cortical actin. We have previously shown that although MYXV lacks an F11 homolog, the F11L gene can be introduced into MYXV promoting the spread of this Leporipoxvirus in natural host cells. Here we show that the F11-encoding (F11L⁺) MYXV strain replicates to higher levels in a number of human cancer cells. We also show that F11L⁺ MYXV induces better tumor control and prolonged survival of mice bearing MDA-MB-231 cancer cells. Furthermore, we show that this virus also spreads more efficiently from the site of growth in one injected tumor, to a second untreated tumor.While we focused mostly on the use of a modified MYXV we were able to show that the effects of F11 on MYXV growth in cancer cells could be mimicked through the use of pharmacological inhibition or siRNA-mediated silencing of key regulators of cortical actin (RhoA, RhoC, mDia1, or LIMK2). These data suggest that it may be possible to increase the oncolytic efficacy of wild-type MYXV using chemical inhibitors of RhoA/C or their downstream targets. Furthermore, since all viruses must overcome barriers to exit posed by structures like cortical actin, these findings suggest that the oncolytic activity of other viruses may be enhanced through similar strategies.     

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

A 6K-Deletion Variant of Salmonid Alphavirus Is Non-Viable but Can Be Rescued through RNA Recombination

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.     

Impedance Changes and Fibrous Tissue Growth after Cochlear Implantation Are Correlated and Can Be Reduced Using a Dexamethasone Eluting Electrode

Background

The efficiency of cochlear implants (CIs) is affected by postoperative connective tissue growth around the electrode array. This tissue formation is thought to be the cause behind post-operative increases in impedance. Dexamethasone (DEX) eluting CIs may reduce fibrous tissue growth around the electrode array subsequently moderating elevations in impedance of the electrode contacts.

Methods

For this study, DEX was incorporated into the silicone of the CI electrode arrays at 1% and 10% (w/w) concentration. Electrodes prepared by the same process but without dexamethasone served as controls. All electrodes were implanted into guinea pig cochleae though the round window membrane approach. Potential additive or synergistic effects of electrical stimulation (60 minutes) were investigated by measuring impedances before and after stimulation (days 0, 7, 28, 56 and 91). Acoustically evoked auditory brainstem responses were recorded before and after CI insertion as well as on experimental days 7, 28, 56, and 91. Additionally, histology performed on epoxy embedded samples enabled measurement of the area of scala tympani occupied with fibrous tissue.

Results

In all experimental groups, the highest levels of fibrous tissue were detected in the basal region of the cochlea in vicinity to the round window niche. Both DEX concentrations, 10% and 1% (w/w), significantly reduced fibrosis around the electrode array of the CI. Following 3 months of implantation impedance levels in both DEX-eluting groups were significantly lower compared to the control group, the 10% group producing a greater effect. The same effects were observed before and after electrical stimulation.

Conclusion

To our knowledge, this is the first study to demonstrate a correlation between the extent of new tissue growth around the electrode and impedance changes after cochlear implantation. We conclude that DEX-eluting CIs are a means to reduce this tissue reaction and improve the functional benefits of the implant by attenuating electrode impedance.     

Can Ecological Receptors Really Be At Risk?

Birds and mammals at hazardous waste sites are routinely modeled for their potential to display harmful toxicological effects as a result of their exposures to contaminants. Although standard desktop measures of the potential for toxicological endpoints to be reached commonly suggest that these receptors should be at ill health (e.g., experiencing reproductive impairment), evidence is lacking from the field that such effects are occurring. Plausibly, the excessive time lapse (frequently several decades) from contaminant release event to site ecological risk assessment, can explain the disconnect. Aided by their relatively brief life spans, these modeled ecological receptors have produced multiple generations to date and may have outbred the chemical stress. In light of the absence of observed health impacts in birds and mammals since the creation of the U.S. Environmental Protection Agency's Superfund program, there may be no need to continue assessing the health of these species. Additionally, given the very few reported instances of observed health effects at terrestrial sites, it may be prudent to replace risk assessment with impact assessment as the essence of a revamped ecological assessment process.     

Altruism Can Be Assessed Correctly Based on Impression

Detection of genuine altruists could be a solution to the problem of subtle cheating. Brown et al. (Evol Psychol 1:42–69, 2003) found that humans could detect altruists using nonverbal cues. However, their experiments can be improved upon in several ways, and further investigation is needed to determine whether altruist-detection abilities are human universals. In our experiment, we used video clips of natural conversations as the stimulus. We asked a sample of Japanese undergraduates to rate their own level of altruism and then to estimate the videotaped targets’ altruism using the same scale. The perceivers were able to estimate the targets’ altruism levels accurately. Perceivers’ altruism score did not affect their ability to discriminate between altruists and non-altruists. Perceivers’ impressions of the altruist and non-altruist targets were also found to be different. Coding of nonverbal behavior of the targets revealed that altruists exhibited more “felt smiles” than non-altruists, which also supports the results of the previous study.     

Testing Genetic Association by Regressing Genotype over Multiple Phenotypes

Complex disorders are typically characterized by multiple phenotypes. Analyzing these phenotypes jointly is expected to be more powerful than dealing with one of them at a time. A recent approach (O''Reilly et al. 2012) is to regress the genotype at a SNP marker on multiple phenotypes and apply the proportional odds model. In the current research, we introduce an explicit expression for the score test statistic and its non-centrality parameter that determines its power. Same simulation studies as those reported in Galesloot et al. (2014) were conducted to assess its performance. We demonstrate by theoretical arguments and simulation studies that, despite its potential usefulness for multiple phenotypes, the proportional odds model method can be less powerful than regular methods for univariate traits. We also introduce an implementation of the proposed score statistic in an R package named iGasso.     

Genetic Mapping of Novel Loci Affecting Canine Blood Phenotypes

Since the publication of the dog genome and the construction of high-quality genome-wide SNP arrays, thousands of dogs have been genotyped for disease studies. For many of these dogs, additional clinical phenotypes are available, such as hematological and clinical chemistry results collected during routine veterinary care. Little is known about the genetic basis of variation in blood phenotypes, but this variation may play an important role in the etiology and progression of many diseases. From a cohort of dogs that had been previously genotyped on a semi-custom Illumina CanineHD array for various genome-wide association studies (GWAS) at Cornell University Hospital for Animals, we chose 353 clinically healthy, adult dogs for our analysis of clinical pathologic test results (14 hematological tests and 25 clinical chemistry tests). After correcting for age, body weight and sex, genetic associations were identified for amylase, segmented neutrophils, urea nitrogen, glucose, and mean corpuscular hemoglobin. Additionally, a strong genetic association (P = 8.1×10⁻¹³) was evident between a region of canine chromosome 13 (CFA13) and alanine aminotransferase (ALT), explaining 23% of the variation in ALT levels. This region of CFA13 encompasses the GPT gene that encodes the transferase. Dogs homozygous for the derived allele exhibit lower ALT activity, making increased ALT activity a less useful marker of hepatic injury in these individuals. Overall, these associations provide a roadmap for identifying causal variants that could improve interpretation of clinical blood tests and understanding of genetic risk factors associated with diseases such as canine diabetes and anemia, and demonstrate the utility of holistic phenotyping of dogs genotyped for disease mapping studies.     

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.     

Prion Protein Peptide Neurotoxicity Can Be Mediated by Astrocytes

A peptide based on amino acids 106-126 of the sequence of human prion protein (PrP106-126) is neurotoxic in culture. A role for astrocytes mediating PrP106-126 toxicity was investigated. The toxicity of PrP106-126 to cerebellar cell cultures was reduced by aminoadipate, a gliotoxin. Normally, PrP106-126 is not toxic to cultures containing neurones deficient in the cellular isoform of prion protein (PrPc). However, PrP106-126 was toxic to cerebellar cells derived from Prnp(0/0) mice (deficient in PrPc expression) when those cerebellar cells were cocultured with astrocytes. This toxicity was found to occur only in the presence of PrPc-positive astrocytes and to be mediated by glutamate. Furthermore, PrPc-positive astrocytes were shown to protect Prnp(0/0) cerebellar cells from glutamate toxicity. This effect could be inhibited by PrP106-126. PrP106-126 did not enhance the toxicity of glutamate to neurones directly. When cerebellar cells were cocultured with astrocytes, the neurones became dependent on astrocytes for protection from glutamate toxicity and expressed an increased sensitivity to glutamate. In such a system, the protective effects of astrocytes against glutamate toxicity to neurones were inhibited by PrP106-126, resulting in a greater reduction in neuronal survival than would have been caused by PrP106-126 when astrocytes were not present. This new model provides a possible mechanism by which the gliosis in prion disease may accelerate the neurodegeneration seen in the later stages of the disease.     

Autophagy Can Be a Killer Even in Apoptosis-Competent Cells

Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.

Addendum to:

Role of Phosphoinositide 3-Kinase in the Autophagic Death of Serum-Deprived PC12 Cells.

A. Guillon-Munos, M.X.P. van Bemmelen and P.G.H. Clarke

Apoptosis 2005; 10:1031-41.     

Democrats and Republicans Can Be Differentiated from Their Faces

Background

Individuals'' faces communicate a great deal of information about them. Although some of this information tends to be perceptually obvious (such as race and sex), much of it is perceptually ambiguous, without clear or obvious visual cues.

Methodology/Principal Findings

Here we found that individuals'' political affiliations could be accurately discerned from their faces. In Study 1, perceivers were able to accurately distinguish whether U.S. Senate candidates were either Democrats or Republicans based on photos of their faces. Study 2 showed that these effects extended to Democrat and Republican college students, based on their senior yearbook photos. Study 3 then showed that these judgments were related to differences in perceived traits among the Democrat and Republican faces. Republicans were perceived as more powerful than Democrats. Moreover, as individual targets were perceived to be more powerful, they were more likely to be perceived as Republicans by others. Similarly, as individual targets were perceived to be warmer, they were more likely to be perceived as Democrats.

Conclusions/Significance

These data suggest that perceivers'' beliefs about who is a Democrat and Republican may be based on perceptions of traits stereotypically associated with the two political parties and that, indeed, the guidance of these stereotypes may lead to categorizations of others'' political affiliations at rates significantly more accurate than chance guessing.     

Imidazoleacetic Acid, a γ-Aminobutyric Acid Receptor Agonist, Can Be Formed in Rat Brain by Oxidation of Histamine

Abstract: It is generally accepted that in mammalian brain histamine is metabolized solely by histamine methyltransferase (HMT), to form tele -methylhistamine, then oxidized to tele -methylimidazoleacetic acid. However, histamine's oxidative metabolite in the periphery, imidazoleacetic acid (IAA), is also present in brain and CSF, and its levels in brain increase after inhibition of HMT. To reinvestigate if brain has the capacity to oxidize histamine and form IAA, conscious rats were injected with [³H]histamine (10 ng), either into the lateral ventricles or cisterna magna, and decapitated 30 min later. In brains of saline-treated rats, most radioactivity recovered was due to tele -methylhistamine and tele -methylimidazoleacetic acid. However, significant amounts of tritiated IAA and its metabolites, IAA-ribotide and IAA-riboside, were consistently recovered. In rats pretreated with metoprine, an inhibitor of HMT, labeled IAA and its metabolites usually comprised the majority of histamine's tritiated metabolites. [³H]Histamine given intracisternally produced only trace amounts of oxidative metabolites. Formation of IAA, a potent GABA-A agonist with numerous neurochemical and behavioral effects, from minute quantities of histamine in brain indicates a need for reevaluation of histamine's metabolic pathway or pathways in brain and suggests a novel mechanism for interactions between histamine and the GABAergic system.