Solubility of neurofibrillary tangles and ultrastructure of paired helical filaments in sodium dodecylsulphate

Summary Temporal cortex from 14 cases of Alzheimer-type dementia and 6 cases of Down's syndrome, all selected for severe Alzheimer pathology, was homogenised in distilled water, NaOH, or sodium dodecylsulphate (SDS) containing 0.1% -mercaptoethanol. The homogenates were stained with Congo red, and the neurofibrillary tangles and plaque cores were counted under crossed-polarisation microscopy. The number of tangles and plaque cores in the water-treated extracts was not related to age, sex, postmortem interval or duration of dementia. The number of tangles after extraction in SDS or NaOH, as a percentage of tangles in water-treated extracts, was 57±25 (mean±SD) for 1% SDS, 43±17 for 5% SDS and 37±22 for 0.2 M NaOH. Plaque cores were essentially insoluble in all three agents. The percentage of tangles insoluble in 1% SDS did not correlated with age or post-mortem interval but decreased with increasing duration of dementia. Enhanced tangle solubility with increasing duration of dementia suggests that the nature of tangles changes with time; one possibility is that this reflects transformation of intracellular to extracellular tangles. Paired helical filament (PHF) length and the number of repeats per PHF were measured in electron micrographs of PHF prepared with and without treatment by 1% SDS. There was no significant multimodality of PHF length to suggest that PHF broke at regular intervals. The mean repeat length (PHF length/number of repeats) was greater for PHF isolated in the presence of 1% SDS than in its absence, showing that SDS affects ultrastructure by untwisting PHF. An untwisting process may also occur in vivo producing the straight filaments found, together with PHF, in tangles and neurites.Supported by Miss E. Buchan (to the MRC Brain Metabolism Unit) and the British Foundation for Age Research and the Wellcome Trust (to P. A. M. Eagles). S. Hussey was in receipt of an MRC Partnership Award.     

Highly diverse heparan sulfate analogue libraries: a novel resource for bioactivity screening of proteins

Introduction Fibroblast growth factors (FGFs) are a multipotent family of growth factors that are important for many biological processes, including development and wound healing. Normal, protease sensitive, prion protein (PrPC) can be converted to the protease resistant, infectious, form (PrPSc) believed to be associated with the pathogenesis of transmissible spongiform encephalopathies. FGFs signal through a family of tyrosine kinase receptors, the FGF receptors (FGFR) with the aid of heparan sulfate (HS), while the role of HS in the biology of PrPC is currently unknown, although depleting cells of HS can prevent production of PrPSc. HS, or its more highly sulfated relation heparin, can exert both positive and negative regulatory activities on a particular FGF‐FGFR combination. The nature of this regulation is determined by the structure of the HS that binds to the proteins. This structure is at least partially determined by the presence of particular sulfate groups along the sugar backbone. Identification of specific sulfate groups that can regulate the activity of proteins has been a long‐term goal in the field. Previously, heparins that had been completely lacking sulfates at specific positions were used to determine the binding and activity requirements for a particular protein. However, this may not necessarily allow for a full examination of the regulatory properties of HS. Here, we present a heparan sulfate analogue library produced by the partial, combinatorial desulfation of heparin. This library was the used to examine the structural properties of heparin required for FGF‐1 signalling through FGFR2c as well as the interactions of HS with PrPC. Materials and methods Porcine intestinal mucosal heparin was subjected to chemical desulfation and enzymatic cleavage. Polysaccharides and oligosaccharides derived from gel filtration chromatography and ion exchange chromatography were tested for their ability to activate FGF signalling through FGF receptors using a BaF3 assay system. Optical biosensors and cell assays were used to study the interaction of PrPC with chemically modified heparin. Results This library possessed vastly more heterogeneity than tissue heparan sulfates, allowing for more systematic screening to help identify those minimal structural features associated with activity. This library was used to examine the different structural features in heparin that support FGF‐1 signalling through FGFR2, showing that HS activity was not strictly dependant on size or charge. In addition, small, low‐sulfated heparins were found to interfere with the PrPC–heparin interaction. Discussion This supports the idea that overall structural features of the HS, rather than just the presence or absence of specific sulfate groups is important for the regulation of protein activity. Future efforts will be focused on further subfractionating the library and identifying specific structural features in HS that support FGF‐1 activity through FGFR2 and other FGFRs as well as the role of HS in the normal function of prion diseases, which may allow for the generation of novel, heparin‐based, therapeutics.     

Postmorten changes in the chemistry and histology of normal and edematous brains.

The brains of 18 patients were examined post mortem for histologic criteria of edema, and samples of white and gray matter were analyzed for water, sodium, and potassium content. In a parallel experimental study, brains of cats with unilateral freezing lesions and resulting cerebral edema were similarly examined immediately after death and up to 18 hours post mortem. In both types of material, in gray matter there was a relatively rapid (within less than 4 hours) increase in water and sodium content and fall in potassium content. In normal and edematous white matter, little change was observed post mortem. No correlation could be demonstrated in any of the material studied between water content and histologic grading for cerebral edema. It is concluded that determination of water content in the white matter postmortem could be a useful tool for the neuropathologist. Histologic assessment of cerebral edema is of little value.     

T cell-mediated immunity in the lung: a Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice.

T cells are important in systemic anticryptococcal defenses, but a role in controlling an initial pulmonary infection has not been demonstrated. A murine model with intratracheal inoculation was developed to study the acquisition and expression of pulmonary T cell-mediated immunity against Cryptococcus neoformans. Infections with four strains of C. neoformans (305, 68A, 613D, and 52D) in two strains of mice (BALB/c and C57BL/6) were examined. Unencapsulated strain 305 and slowly growing strain 68A were readily controlled apparently by nonimmune pulmonary defenses, and no extrapulmonary dissemination was detected. Strain 613D grew progressively in the lungs and disseminated to the brain and spleen. Strain 52D initially grew rapidly in the lungs and disseminated to the spleen, but a clearance mechanism developed in the lungs after day 7 postinfection and in the spleen after day 28. SCID and athymic nude mice were unable to clear a strain 52D pulmonary infection, and a lethal disseminated infection occurred. Pulmonary clearance could be adoptively transferred into SCID mice infected with strain 52D by use of immune T cells from the spleen and lungs and hilar lymph nodes of infected immunocompetent donors. Furthermore, pulmonary clearance was almost 100-fold better in SCID mice that received immune T cells from the lungs and hilar lymph nodes than in those that received immune T cells from the spleen, even though equivalent levels of delayed-type hypersensitivity were transferred by both cell populations. These adoptive transfer studies suggested that the lung and hilar lymph node T cells from immune animals either are enriched in such a way as to mediate protective immunity or home to the lungs better than do splenic T cells.     

Replication of a type I avian adenovirus (CELO) mutant in the chicken embryo

Summary Replication of a CELO large plaque (LP) mutant and that of its wild type small plaque (SP) parent was studied in the chorioallantoic membrane (CAM) and in the amnion of 11-day-old embryos. Although both strains produced essentially the same amount of virus in the tissue fluids, they differed in their rates of replication. Replication of the SP parent was maximal in the CAM 24 to 48 hours before that of the LP mutant. Whereas inclusions were observed in SP inoculated CAM 48 hours PI and were present during the course of study; LP inclusions were rare at 72 hours PI and thereafter; LP inclusions were seen at 72 hours PI. Fewer SP than LP particles were required to produce inclusions. No inclusions were seen in sections of the trachea and liver removed at 96 hours PI from embryos inoculated via the amniotic sac with LP and SP virus.Contribution 1466 of the Rhode Island Agriculture Experimental Station.     

A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension

Kinetochores play an essential role in chromosome segregation by forming dynamic connections with spindle microtubules. Here, we identify a set of 10 copurifying kinetochore proteins from Caenorhabditis elegans, seven of which were previously uncharacterized. Using in vivo assays to monitor chromosome segregation, kinetochore assembly, and the mechanical stability of chromosome-microtubule attachments, we show that this copurifying protein network plays a central role at the kinetochore-microtubule interface. In addition, our analysis suggests that the network is comprised of three groups of proteins that contribute in distinct ways to this interface: KNL proteins act after the assembly of centromeric chromatin to generate the core of the microtubule-binding interface, MIS proteins control the rate and extent of formation of this interface, and NDC proteins are necessary to sustain tension during interactions with spindle microtubules. We also purify a similar set of associated proteins from human cells that includes four novel proteins and has recognizable homologs from each functional class. Thus, this protein network is a conserved constituent of the outer kinetochore, and the functions defined by our analysis in C. elegans are likely to be widely relevant.     

Athletic Medical Insurance Practices at NCAA Division I Institutions

As the cost of athletic medical insurance continues to rise, athletic departments are searching for ways to continue to provide quality insurance coverage while keeping costs contained. There are few published articles dealing with the specific topic of the daily operations of maintaining an athletic medical insurance program. We sent an 18-item questionnaire to the 295 active NCAA Division I head athletic trainers to ascertain the current trends in athletic medical insurance. Of these, 207 (70%) responded. Of the respondents, 85% were primarily responsible for the administration of their athletic department's insurance coverage, although they had received no formal training in insurance management. Most athletic departments carry secondary policies and many report having a deductible. A wide range of insurance coverage and premiums were reported.     

Response of vestibular neurons to head rotations in vertical planes. III. Response of vestibulocollic neurons to vestibular and neck stimulation

1. To compare the properties of the vestibulocollic reflex (VCR) with those of vestibular neurons projecting to the neck [vestibulocollic (VC) neurons], we have studied the behavior of the latter in the decerebrate cat. Neurons were identified by their antidromic responses to stimulation in C1-C2, but not C5. Responses to stimulation of vestibular and neck receptors were produced by rotation of the body and head in vertical planes. 2. We determined the plane of whole body (vestibular) or body with head counter-rotated (neck) rotation, which produced the maximal modulation of each neuron (response vector orientation). Neuron dynamics were then studied with sinusoidal (0.02-2 Hz) stimuli aligned with this orientation. 3. On the basis of dynamics and vector orientation, the neuron was assigned a vestibular input classification of otolith, vertical canal, otolith + canal, or spatial-temporal convergence (STC). 4. The properties of this sample of VC neurons are similar to those of a larger population of vestibular neurons whose projection was not identified. For example, the distributions of cells with different types of vestibular inputs were roughly the same; in particular, few cells showed STC responses. In addition, there was no evidence of significant convergence of like canals across the midline (e.g., right anterior + left anterior). 5. Also similar to the larger unidentified population, 80% of VC neurons tested for neck input received such an input. The neck and vestibular responses tended to be antagonistic; the vector orientations were usually opposite, and the response gains and phases similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Instability of normal (CTG)n alleles in the DM kinase gene.

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.