Analysis of the apparent biphasic axonal transport kinetics of fucosylated glycoproteins

Following intraocular injection of [3H]fucose, which labels many glycoproteins of retinal ganglion cells, the accumulation of transported radioactivity arriving at the superior colliculus (nerve terminals) peaks within a few hours and decays with a time course of hours. Then, over a period of several days, radioactivity again accumulates at the superior colliculus and then decays with a half-life of days. The second peak also represents fast transported material since it occurs almost simultaneously along the optic nerve and tract as well as at the nerve endings. Such data have been interpreted as evidence for both a group of rapidly released, rapidly transported glycoproteins (first peak) and a group of slowly released but rapidly transported glycoproteins (second peak). We investigated this supposition by studying in more detail the metabolism of some individual fucosylated proteins in both the retina and superior colliculus. We noted that much of the radioactivity incorporated in fucosylated glycoproteins at the retina was rapidly metabolized (with a turnover on the order of hours), while the remainder of the fucosylated moieties had a metabolic half-life on the order of days. This was also true of the metabolic behavior of several individual glycoproteins, selected for study because they are major components of the group committed to transport and accumulating in two waves at the superior colliculus. In other experiments we injected [35S]methionine intraocularly and examined the metabolism in the retina and the kinetics of transport to the superior colliculus of the peptide backbone of these same individual proteins. In contrast to the two waves of accumulation of radioactivity from [3H]fucose, accumulation of radioactivity of the peptide backbone of the same glycoproteins was monophasic. Our explanation of these data involves the presence of two types of fucose moieties on the peptides. One group of fucose moieties is labile and is lost from the peptide backbone over a period of hours. Other fucose moieties are approximately as metabolically stable as the peptide backbones to which they are attached. The actual peptide backbones of the glycoproteins are committed to rapid transport over a period of several days. Thus, the first (and most prominent) peak of transported radioactivity in [3H]fucosylated glycoproteins does not represent a discrete phase of transport but, rather, is the summation of kinetics of gradual arrival of proteins and the rapid drop in their specific radioactivity as the more labile moieties of [3H]fucose are lost.( TRUNCATED AT 400 WORDS

Fate of myelin lipids during degeneration and regeneration of peripheral nerve: an autoradiographic study

Four weeks after labeling myelin lipids with an intraneural injection of 3H-acetate, sciatic nerves were crushed, and the distribution of radiolabeled myelin lipids was followed by autoradiography from 1 d to 10 weeks later. Just prior to crush, silver grains were localized to the myelin sheath. Three days after crush, axons were degenerating and myelin sheaths were breaking down; silver grains appeared over lipid droplets within Schwann cells, fibroblasts, and macrophages. One week after crush the basal-lamina-delimited Schwann-cell tubes (Bungner bands) contained myelin debris, and some tubes already contained regenerating axons. Schwann cells were often displaced to the periphery of the tubes by phagocytes containing heavily labeled myelin debris; extratubal macrophages within the endoneurium contained labeled lipid droplets but no myelin debris. Two weeks after nerve crush silver grains were associated with newly formed myelin around regenerating axons. Many extratubal endoneurial macrophages now contained labeled myelin debris and lipid droplets. By 3 weeks myelination of regenerating axons was advanced, and the myelin sheaths were well labeled. Extratubal macrophages had become the major labeled structure within the nerve because they contained large amounts of labeled myelin debris and lipid droplets. From 4 to 10 weeks after nerve crush the new myelin sheaths continued to thicken and to be well labeled. Debris- laden extratubal macrophages remained the major site of labeled material within the endoneurium. Our results confirm that there is reutilization of myelin cholesterol by Schwann cells to form new myelin, and indicate that some lipid catabolism takes place in Schwann cells and endoneurial fibroblasts prior to infiltration of the nerve by macrophages. However, most of the myelin debris is phagocytized by macrophages within 1–2 weeks following nerve injury. These debris-laden macrophages persist within the nerve for many weeks, indicating that much of the salvaged cholesterol is not reutilized for myelin regeneration

A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγcnull-humanized mouse model. The UL133-UL138NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations

Validity and worth in the science curriculum: learning school science outside the laboratory

It is widely acknowledged that there are problems with school science in many developed countries of the world. Such problems manifest themselves in a progressive decline in pupil enthusiasm for school science across the secondary age range and the fact that fewer students are choosing to study the physical sciences at higher levels and as careers. Responses to these developments have included proposals to reform the curriculum, pedagogy and the nature of pupil discussion in science lessons. We support such changes but argue from a consideration of the aims of science education that secondary school science is too rooted in the science laboratory; substantially greater use needs to be made of out-of-school sites for the teaching of science. Such usage should result in a school science education that is more valid and more motivating and is better at fulfilling defensible aims of school science education. Our contention is that laboratory-based school science teaching needs to be complemented by out-of-school science learning that draws on the actual world (e.g. through fieldtrips), the presented world (e.g. in science centres, botanic gardens, zoos and science museums) and the virtual worlds that are increasingly available through information and communications technologies (ICT)

Primary demyelination induced by exposure to tellurium alters Schwann cell gene expression: a model for intracellular targeting of NGF receptor

Exposure of developing rats to tellurium results in a highly synchronous segmental demyelination of peripheral nerves with sparing of axons; this demyelination is followed closely by a period of rapid remyelination. Demyelination occurs subsequent to a tellurium-induced block in the synthesis of cholesterol, the major myelin lipid. We utilized the techniques of Northern blotting, in situ hybridization, and immunocytochemistry to examine temporal alterations in Schwann cell gene expression related to demyelination and remyelination. Tellurium- induced demyelination is associated with downregulation of myelin protein expression and a corresponding upregulation of NGF receptor (NGF-R) and glial fibrillary acidic protein (GFAP) expression. Steady- state mRNA levels (expressed on a “per nerve” basis) for P0, the major myelin protein, were decreased by about 50% after 5 d of tellurium exposure, while levels of mRNA for NGF-R and GFAP were markedly increased (about 15-fold). In situ hybridization of teased fibers suggested that the increase in steady-state mRNA levels for NGF-R was primarily associated with demyelinated internodes and not with adjacent unaffected internodes. Although P0 message was almost totally absent from demyelinating internodes, it was also reduced in normal-appearing internodes as well. This suggests that limiting the supply of a required membrane component (cholesterol) may lead to partial downregulation of myelin gene expression in all myelinating Schwann cells. In partially demyelinated internodes, NGF-R and GFAP immunofluorescence appeared largely confined to the demyelinated regions. This suggests specific targeting of these proteins to local areas of the Schwann cell where there is myelin loss. These results demonstrate that demyelination is associated with reversion of the affected Schwann cells to a precursor cell phenotype. Because axons remain intact, our results suggest that these changes in Schwann cell gene expression do not require input from a degenerating axon, but instead may depend on whether concerted synthesis of myelin is occurring

Automatic Identification of Defects on Eggshell Through a Multispectral Vision System

The objective of this research was to develop an off-line artificial vision system to automatically detect defective eggshells, i.e., dirty or cracked eggshells, by employing multispectral images with the final purpose to adapt the system to an on-line grading machine. In particular, this work was focused to study the feasibility of identifying organic stains on brown eggshells (dirty eggshell), caused by blood, feathers, feces, etc., from natural stains, caused by deposits of pigments on the outer layer of clean eggshells. During the analysis a total of 384 eggs were evaluated (clean: 148, dirty: 236). Dirty samples were evaluated visually in order to classify them according to the kind of defect (blood, feathers, and white, clear or dark feces), and clean eggshells were classified on the basis of the colour of the natural stains (clear or dark). For each sample digital images were acquired by employing a Charged Coupled Device (CCD) camera endowed with 15 monochromatic filters (440-940 nm). A Matlab® function was developed in order to automate the process and analyze images, with the aim to classify samples as clean or dirty. The program was constituted by three major steps: first, the research of an opportune combination of monochromatic images in order to isolate the eggshell from the background; second, the detection of the dirt stains; third, the classification of the images samples into the dirty or clean group on the basis of geometric characteristics of the stains (area in pixel). The proposed classification algorithm was able to correctly classify near 98% of the samples with a very low processing time (0.05s). The robustness of the proposed classification was observed applying an external validation to a second set of samples (n = 178), obtaining similar percentage of correctly classified samples (97%)

Learning to Teach About Ideas and Evidence in Science : The Student Teacher as Change Agent

A collaborative curriculum development project was set up to address the lack of good examples of teaching about ideas and evidence and the nature of science encountered by student teachers training to teach in the age range 11-16 in schools in England. Student and teacher-mentor pairs devised, taught and evaluated novel lessons and approaches. The project design required increasing levels of critique through cycles of teaching, evaluation and revision of lessons. Data were gathered from interviews and students' reports to assess the impact of the project on student teachers and to what extent any influences survived when they gained their first teaching posts. A significant outcome was the perception of teaching shifting from the delivery of standard lessons in prescribed ways to endeavours demanding creativity and decision-making. Although school-based factors limited newly qualified teachers' chances to use new lessons and approaches and therefore act as change-agents in schools, the ability to critique curriculum materials and the recognition of the need to create space for professional dialogue were durable gains

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells.

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CDCD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo

The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection