Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.

A T cell-mediated immune response is initiated by the T cell receptor (TCR) interacting with peptide-bound MHC (pMHC) on an infected cell. The mechanism by which this interaction triggers intracellular phosphorylation of the TCR, which lacks a kinase domain, remains poorly understood. Here, we have introduced the TCR and associated signalling molecules into a nonimmune cell and reconstituted ligand-specific signalling when these cells are conjugated with antigen presenting cells. We show that signalling requires the differential segregation of a phosphatase and kinase in the plasma membrane. An artificial, chemically-controlled receptor system generates the same effect as TCR-pMHC, demonstrating that the binding energy of an extracellular protein-protein interaction can drive the spatial segregation of membrane proteins without a transmembrane conformational change. This general mechanism may extend to other receptors that rely on extrinsic kinases, including, as we demonstrate, chimaeric antigen receptors being developed for cancer immunotherapy.

Background

Dust exposure is a well-known occupational hazard for terrestrial workers and astronauts alike and will continue to be a concern as humankind pursues exploration and habitation of objects beyond Earth. Humankind’s limited exploration experience with the Apollo Program indicates that exposure to dust will be unavoidable. Therefore, NASA must assess potential toxicity and recommend appropriate mitigation measures to ensure that explorers are adequately protected. Visual acuity is critical during exploration activities and operations aboard spacecraft. Therefore, the present research was performed to ascertain the ocular toxicity of authentic lunar dust.

Methods

Small (mean particle diameter = 2.9 ± 1.0 μm), reactive lunar dust particles were produced by grinding bulk dust under ultrapure nitrogen conditions. Chemical reactivity and cytotoxicity testing were performed using the commercially available EpiOcularTM assay. Subsequent in vivo Draize testing utilized a larger size fraction of unground lunar dust that is more relevant to ocular exposures (particles <120 μm; median particle diameter = 50.9 ± 19.8 μm).

Results

In vitro testing indicated minimal irritancy potential based on the time required to reduce cell viability by 50% (ET50). Follow-up testing using the Draize standard protocol confirmed that the lunar dust was minimally irritating. Minor irritation of the upper eyelids was noted at the 1-hour observation point, but these effects resolved within 24 hours. In addition, no corneal scratching was observed using fluorescein stain.

Conclusions

Low-titanium mare lunar dust is minimally irritating to the eyes and is considered a nuisance dust for ocular exposure. No special precautions are recommended to protect against ocular exposures, but fully shielded goggles may be used if dust becomes a nuisance.

Canine Hip Dysplasia (CHD) is a common, painful and debilitating orthopaedic disorder of dogs with a partly genetic, multifactorial aetiology. Worldwide, potential breeding dogs are evaluated for CHD using radiographically based screening schemes such as the nine ordinally-scored British Veterinary Association Hip Traits (BVAHTs). The effectiveness of selective breeding based on screening results requires that a significant proportion of the phenotypic variation is caused by the presence of favourable alleles segregating in the population. This proportion, heritability, was measured in a cohort of 13,124 Australian German Shepherd Dogs born between 1976 and 2005, displaying phenotypic variation for BVAHTs, using ordinal, linear and binary mixed models fitted by a Restricted Maximum Likelihood method. Heritability estimates for the nine BVAHTs ranged from 0.14–0.24 (ordinal models), 0.14–0.25 (linear models) and 0.12–0.40 (binary models). Heritability for the summed BVAHT phenotype was 0.30±0.02. The presence of heritable variation demonstrates that selection based on BVAHTs has the potential to improve BVAHT scores in the population. Assuming a genetic correlation between BVAHT scores and CHD-related pain and dysfunction, the welfare of Australian German Shepherds can be improved by continuing to consider BVAHT scores in the selection of breeding dogs, but that as heritability values are only moderate in magnitude the accuracy, and effectiveness, of selection could be improved by the use of Estimated Breeding Values in preference to solely phenotype based selection of breeding animals.

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr531) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.

The nucleolus is the subnuclear organelle responsible for ribosome subunit biogenesis and can also act as a stress sensor. It forms around clusters of ribosomal DNA (rDNA) and is mainly organised in three subcompartments, i.e. fibrillar centre, dense fibrillar component and granular component. Here, we describe the localisation of 21 protein factors to an intranucleolar region different to these main subcompartments, called the intranucleolar body (INB). These factors include proteins involved in DNA maintenance, protein turnover, RNA metabolism, chromatin organisation and the post-translational modifiers SUMO1 and SUMO2/3. Increase in the size and number of INBs is promoted by specific types of DNA damage and depends on the functional integrity of the nucleolus. INBs are abundant in nucleoli of unstressed cells during S phase and localise in close proximity to rDNA with heterochromatic features. The data suggest the INB is linked with regulation of rDNA transcription and/or maintenance of rDNA.

Electronic supplementary material

The online version of this article (doi:10.1007/s00412-011-0327-8) contains supplementary material, which is available to authorized users.

Acute chemotherapy-related leukoencephalopathy can present similar to acute stroke with symptoms including aphasia, dysarthria, and hemiplegia. Differentiation based on clinical appearance is challenging, and physicians must distinguish between the 2 conditions rapidly to institute appropriate therapies. An 8-year-old male with acute lymphoblastic leukemia receiving chemotherapy, including intrathecal methotrexate, presented to our emergency center with 2 hours of expressive aphasia and flaccid right hemiplegia. Emergent magnetic resonance imaging (MRI) was obtained, demonstrating diffusion restriction within bilateral corona radiata and centrum semiovale. Magnetic resonance perfusion revealed mildly increased perfusion, a finding inconsistent with ischemic stroke and previously unreported in acute chemotherapy-related leukoencephalopathy without necrosis. This increased perfusion conclusively eliminated stroke from the clinical differential. Magnetic resonance perfusion imaging proved valuable to rapidly distinguish acute chemotherapy-related leukoencephalopathy from ischemia, and the evaluation of perfusion alterations in this disorder may provide further insight into the pathophysiology of this entity.

Abstract

We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.

FRET analysis of cell lines expressing fluorescently tagged histones on separate nucleosomes demonstrates that variations in chromosome compaction occur during mitosis.

We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLaH2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.

The p53-inducible BH3-only protein PUMA is a key mediator of p53-dependent apoptosis, and PUMA has been shown to function by activating Bax and mitochondrial outer membrane permeabilization. In this study we describe an ability of PUMA to induce autophagy that leads to the selective removal of mitochondria. This function of PUMA depends on Bax/Bak and can be reproduced by overexpression of Bax. The induction of autophagy coincides with cytochrome c release, and taken together the results suggest that PUMA functions through Bax to induce mitochondrial autophagy in response to mitochondrial perturbations. Surprisingly, inhibition of PUMA or Bax-induced autophagy dampens the apoptotic response, suggesting that under some circumstances the selective targeting of mitochondria for autophagy can enhance apoptosis.

The Lewis (LEW) strain of rat appears more sensitive to nicotine than other strains in self administration, conditioned place preference, and drug discrimination behavioral studies. The present study sought to further evaluate the behavioral effects of chronic nicotine treatment in the LEW strain by assessing spontaneous activity, which has consistently revealed sensitization to chronic nicotine administration in Sprague Dawley (SD) rats. High active and low active male and female LEW rats (N = 8 per group) were treated twice daily with either nicotine (0.4 mg/kg, sc) or vehicle for 14 consecutive days. Regardless of baseline activity level or sex, spontaneous activity was significantly decreased, compared to saline-treated rats, after a single nicotine injection. However, spontaneous activity increased in both low and high activity rats (both sexes) over the two-weeks of nicotine administration to levels that were significantly higher than saline-treated rats. Based on these findings, acute and chronic nicotine administration had greater suppressive and enhancing effects on spontaneous activity in LEW rats compared to other strains of rats previously studied. These results further clarify the behavioral sensitivity of the LEW strain of rat to nicotine exposure and lend credence to the role of genetics in the individual susceptibility to nicotine dependence.

We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.

A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB3H4 labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB3H4 labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.

The effects of acute nicotine administration (0.09 mg/kg nicotine), chronic nicotine administration (6.3 mg/kg/d nicotine for 14 d), and withdrawal from chronic nicotine administration on fear conditioning in C57BL/6 mice were examined. Mice were trained using two coterminating conditioned stimulus (30 s; 85 dB white noise)– unconditioned stimulus (2 s; 0.57 mA foot shock) pairings and tested 24 h later for contextual and cued fear conditioning. Acute nicotine administration enhanced contextual fear conditioning, chronic nicotine administration had no effect on contextual fear conditioning, and withdrawal from chronic nicotine administration impaired contextual fear conditioning. Plasma nicotine concentrations were similar after acute and chronic treatment and were within the range reported for smokers. During withdrawal, concentrations of nicotine were undetectable. An acute dose of nicotine (0.09 mg/kg) during withdrawal from chronic nicotine treatment reversed withdrawal-associated deficits in contextual fear conditioning. The results suggest that tolerance to the effects of nicotine on contextual fear conditioning develops with chronic nicotine treatment at a physiologically relevant dose, and withdrawal from this chronic nicotine treatment is associated with impairments in contextual fear conditioning. These findings provide a model of how the effects of nicotine on learning may contribute to the development and maintenance of nicotine addiction.

Summary

Background

Glycoprotein VI (GPVI) is a physiological receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerisation of GPVI has been proposed as necessary for the interaction with collagen, although direct evidence of dimerisation has not been reported in cell lines or platelets.

Objectives

To investigate oligomerisation of GPVI in transfected cell lines and in platelets under nonstimulated conditions.

Methods and Results

By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extra-cellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerisation of GPVI under these conditions. A chemical cross-linker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the FcR γ-chain.

Conclusions

The present results directly demonstrate GPVI-FcR γ-chain oligomerisation at the surface of the platelet, and thereby add to the growing evidence that oligomerisation of GPVI may be a pre-requisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

Iron deficiency in children has been associated with behavioural disorder and developmental delay. Screening for iron deficiency was offered to all 527 children aged between one and four years in an inner city practice. Half the children belong to an ethnic minority group, and there is widespread social deprivation in the area. Capillary haemoglobin concentration and mean corpuscular volume were estimated in 365 children (69%). Dietary history, birth weight and current weight were also recorded. Fifty-eight (16%) of the children were iron deficient as defined by a mean corpuscular volume of less than 75 fl and/or a haemoglobin concentration of less than 10.5 g dl-1. All were hypochromic and among 23 tested all had serum ferritin levels below 10µg I-1. Twenty-one children (5.8%) were anaemic (haemoglobin concentration less than 10.5 g dl-1). Anaemia was significantly more common among children who were currently underweight but was not related to weight at birth. Iron deficiency was significantly more prevalent in non-Caucasian children – 25.0% compared with 7.8% of Caucasian children. There was also a significant linear decrease in iron deficiency with increasing age. Sex, weight at birth, current weight, whether breast fed, age weaned or whether on a vegetarian diet were not significant factors in iron deficiency. Iron supplements were given to all the children with iron deficiency.

In view of the high prevalence of iron deficiency, all children in the practice are now routinely offered screening for iron deficiency at the age of 14 months. The programme has been welcomed by all parents. It is suggested that screening for iron deficiency should be part of routine child surveillance.

The delivery of health care has been improved in an inner-city well-baby clinic by staffing the clinic with members of a primary health care team and by offering a therapeutic service together with a preventive service. Over a three-year period attendance at the clinic has doubled and the uptake of immunization has increased. Of children registered with the practice supplying the health care team, 95% are up to date with diphtheria, tetanus and polio immunizations and 93% have been immunized against measles. It is suggested that general practitioners should have greater involvement in the running of well-baby clinics.

A target behavior program, structured within a token economy project, was implemented to modify the behavior of an institutionalized patient who exhibited excessive rates of crying and no smiling responses. To affect both responses concurrently, token costs were made contingent upon crying and token payments and/or social reinforcements were provided for smiling. The results indicated both the feasibility of eliminating “anxiety-depression” within an institutional environment and the efficacy of the treatment procedures 14 months after discharge.