Molecular cloning and functional expression of Xenopus laevis oocyte ATP-activated P2X4 channels

All cells contain mechanosensitive ion channels, yet the molecular identities of most are unknown. The purpose of our study was to determine what encodes the Xenopus oocyte's mechanosensitive cation channel. Based on the idea that homologues to known channels might contribute to the stretch channels, we screened a Xenopus oocyte cDNA library with cation channel probes. Whereas other screens were negative, P2X probes identified six isoforms of the P2X4 subtype of ATP-gated channels. From RNase protection assays and RT-PCR, we demonstrated that Xenopus oocytes express P2X4 mRNA. In expression studies, four isoforms produced functional ATP-gated ion channels; however, one, xP2X4c, had a conserved cysteine replaced by a tyrosine and failed to give rise to functional channels. By changing the tyrosine to a cysteine, we showed that this cysteine was crucial for function. We raised antibodies against a Xenopus P2X4 C-terminal peptide to investigate xP2X4 protein expression. This affinity purified anti-xP2X4 antibody recognized a 56 kDa glycosylated Xenopus P2X4 protein expressed in stably transfected HEK-293 cells and in P2X4 cDNA injected oocytes overexpressing the cloned P2X4 channels; however, it failed to recognize proteins in control, uninjected oocytes. This suggests that P2X4 channels and mechanosensitive cation channels are not linked. Instead, oocyte P2X4 mRNA may be part of the stored pool of stable maternal mRNA that remains untranslated until later developmental stages.     

Analysis of a clonal lineage of HIV-1 envelope V2/V3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors

V2/V3 conformational epitope antibodies that broadly neutralize HIV-1 (PG9 and PG16) have been recently described. Since an elicitation of previously known broadly neutralizing antibodies has proven elusive, the induction of antibodies with such specificity is an important goal for HIV-1 vaccine development. A critical question is which immunogens and vaccine formulations might be used to trigger and drive the development of memory B cell precursors with V2/V3 conformational epitope specificity. In this paper we identified a clonal lineage of four V2/V3 conformational epitope broadly neutralizing antibodies (CH01 to CH04) from an African HIV-1-infected broad neutralizer and inferred their common reverted unmutated ancestor (RUA) antibodies. While conformational epitope antibodies rarely bind recombinant Env monomers, a screen of 32 recombinant envelopes for binding to the CH01 to CH04 antibodies showed monoclonal antibody (MAb) binding to the E.A244 gp120 Env and to chronic Env AE.CM243; MAbs CH01 and CH02 also bound to transmitted/founder Env B.9021. CH01 to CH04 neutralized 38% to 49% of a panel of 91 HIV-1 tier 2 pseudoviruses, while the RUAs neutralized only 16% of HIV-1 isolates. Although the reverted unmutated ancestors showed restricted neutralizing activity, they retained the ability to bind to the E.A244 gp120 HIV-1 envelope with an affinity predicted to trigger B cell development. Thus, E.A244, B.9021, and AE.CM243 Envs are three potential immunogen candidates for studies aimed at defining strategies to induce V2/V3 conformational epitope-specific antibodies.     

Immunogenic glycoconjugates implicated in parasitic nematode diseases

Parasitic nematodes infect billions of people world-wide, often causing chronic infections associated with high morbidity. The greatest interface between the parasite and its host is the cuticle surface, the outer layer of which in many species is covered by a carbohydrate-rich glycocalyx or cuticle surface coat. In addition many nematodes excrete or secrete antigenic glycoconjugates (ES antigens) which can either help to form the glycocalyx or dissipate more extensively into the nematode's environment. The glycocalyx and ES antigens represent the main immunogenic challenge to the host and could therefore be crucial in determining if successful parasitism is established. This review focuses on a few selected model systems where detailed structural data on glycoconjugates have been obtained over the last few years and where this structural information is starting to provide insight into possible molecular functions.     

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS)

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.     

Biosemiosis and the Cellular Basis of Mind

The human brain is a complex organ made up of neurons and several other cell types, and whose role is processing information for use in elicitation of behaviors. To accomplish this, the brain requires large amounts of energy, and this energy is obtained by the oxidation of glucose (Glc). However, the question of how the oxidation of Glc by individual neurons in brain results in their collective ability to rapidly generate feats of cognition that allow them to recognize the nature of the universe in which they live and to communicate this information remains unclear. In this article, insights into this process are provided by first considering the brain’ s homeostatic “operating system” for supply of energy to stimulated neurons, and how this system defines the basic unit of brain “structure”. This is followed by consideration of the brain’s “two-cell” neuronal communication mechanism which defines the basic unit of brain “function”. Finally, an analysis of the nature of frequency-encoded “neuronal languages” that enable ensembles of neurons to translate energy derived from the oxidation of Glc into a collective “mind”, the aggregate of all brain processes including those involving perception, thought, insight, foresight, imagination and behavior.     

Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

“An Impediment to Living Life”: Why and How Should We Measure Stiffness in Polymyalgia Rheumatica?

Objectives

To explore patients’ concepts of stiffness in polymyalgia rheumatica (PMR), and how they think stiffness should be measured.

Methods

Eight focus groups were held at three centres involving 50 patients with current/previous PMR. Each group had at least one facilitator and one rapporteur making field notes. An interview schedule was used to stimulate discussion. Interviews were recorded, transcribed and analysed using an inductive thematic approach.

Results

Major themes identified were: symptoms: pain, stiffness and fatigue; functional impact; impact on daily schedule; and approaches to measurement. The common subtheme for the experience of stiffness was “difficulty in moving”, and usually considered as distinct from the experience of pain, albeit with a variable overlap. Some participants felt stiffness was the “overwhelming” symptom, in that it prevented them carrying out “fundamental activities” and “generally living life”. Diurnal variation in stiffness was generally described in relation to the daily schedule but was not the same as stiffness severity. Some participants suggested measuring stiffness using a numeric rating scale or a Likert scale, while others felt that it was more relevant and straightforward to measure difficulty in performing everyday activities rather than about stiffness itself.

Conclusions

A conceptual model of stiffness in PMR is presented where stiffness is an important part of the patient experience and impacts on their ability to live their lives. Stiffness is closely related to function and often regarded as interchangeable with pain. From the patients’ perspective, visual analogue scales measuring pain and stiffness were not the most useful method for reporting stiffness; participants preferred numerical rating scales, or assessments of function to reflect how stiffness impacts on their daily lives. Assessing function may be a pragmatic solution to difficulties in quantifying stiffness.     

Symmetrical Dose-Dependent DNA-Methylation Profiles in Children with Deletion or Duplication of 7q11.23

Epigenetic dysfunction has been implicated in a growing list of disorders that include cancer, neurodevelopmental disorders, and neurodegeneration. Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders with broad phenotypic spectra caused by deletion and duplication, respectively, of a 1.5-Mb region that includes several genes with a role in epigenetic regulation. We have identified striking differences in DNA methylation across the genome between blood cells from children with WS or Dup7 and blood cells from typically developing (TD) children. Notably, regions that were differentially methylated in both WS and Dup7 displayed a significant and symmetrical gene-dose-dependent effect, such that WS typically showed increased and Dup7 showed decreased DNA methylation. Differentially methylated genes were significantly enriched with genes in pathways involved in neurodevelopment, autism spectrum disorder (ASD) candidate genes, and imprinted genes. Using alignment with ENCODE data, we also found the differentially methylated regions to be enriched with CCCTC-binding factor (CTCF) binding sites. These findings suggest that gene(s) within 7q11.23 alter DNA methylation at specific sites across the genome and result in dose-dependent DNA-methylation profiles in WS and Dup7. Given the extent of DNA-methylation changes and the potential impact on CTCF binding and chromatin regulation, epigenetic mechanisms most likely contribute to the complex neurological phenotypes of WS and Dup7. Our findings highlight the importance of DNA methylation in the pathogenesis of WS and Dup7 and provide molecular mechanisms that are potentially shared by WS, Dup7, and ASD.