mRNA levels of BACE1 and its interacting proteins, RTN3 and PPIL2, correlate in human post mortem brain tissue.

ß-Site amyloid precursor protein cleaving enzyme (BACE1) is the rate-limiting enzyme for production of Aß peptides, proposed to drive the pathological changes found in Alzheimer's disease (AD). Reticulon 3 (RTN3) is a negative modulator of BACE1 (ß-secretase) proteolytic activity, while peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) positively regulated BACE1 gene expression in a cell-based assay. This study aimed to analyze RTN3 and PPIL2 mRNA levels in four brain regions from individuals with AD and controls. BACE1 mRNA had been previously quantified in the samples, as had glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE), to track changing cell populations in the tissue. mRNA levels in the human post mortem brain tissue were assayed using quantitative real-time polymerase chain reaction (qPCR) and qbase(PLUS), employing validated stably expressed reference genes. No differences in RTN3 or PPIL2 mRNA levels were found in individuals with AD, compared to controls. Both RTN3 and PPIL2 mRNA levels correlated significantly with BACE1 mRNA and all three showed similar disease stage-dependent changes with respect to NSE and GFAP. These findings indicated that the in vitro data demonstrating an effect of PPIL2 on BACE1 expression have functional relevance in vivo. Further research into BACE1-interacting proteins could provide a fruitful approach to the modulation of this protease and consequently Aß production.

Platelet beta-secretase activity is increased in Alzheimer's disease.

beta-Secretase activity is the rate-limiting step in Abeta peptide production from amyloid precursor protein. Abeta is a major component of Alzheimer's disease (AD) cortical amyloid plaques. beta-Secretase activity is elevated in post mortem brain tissue in AD. The current study investigated whether beta-secretase activity was also elevated in peripheral blood platelets. We developed a novel fluorimetric beta-secretase activity assay to investigate platelets isolated from individuals with AD (n=86), and age-matched controls (n=115). Platelet membrane beta-secretase activity (expressed as initial rate) varied over fourfold between individuals, raising important questions about in vivo regulation of this proteolytic activity. Nonetheless, we identified a significant 17% increase in platelet membrane beta-secretase activity in individuals with AD compared to controls (p=0.0003, unpaired t-test). Platelet membrane beta-secretase activity did not correlate with mini-mental state examination (MMSE) score in the AD group (mean MMSE=17.7, range 1-23), indicating that the increase did not occur as a secondary result of the disease process, and may even have preceded symptom onset.

An assessment of the impact of the NHS Health Technology Assessment Programme.

OBJECTIVES: To consider how the impact of the NHS Health Technology Assessment (HTA) Programme should be measured. To determine what models are available and their strengths and weaknesses. To assess the impact of the first 10 years of the NHS HTA programme from its inception in 1993 to June 2003 and to identify the factors associated with HTA research that are making an impact. DATA SOURCES: Main electronic databases from 1990 to June 2005. The documentation of the National Coordinating Centre for Health Technology Assessment (NCCHTA). Questionnaires to eligible researchers. Interviews with lead investigators. Case study documentation. REVIEW METHODS: A literature review of research programmes was carried out, the work of the NCCHTA was reviewed, lead researchers were surveyed and 16 detailed case studies were undertaken. Each case study was written up using the payback framework. A cross-case analysis informed the analysis of factors associated with achieving payback. Each case study was scored for impact before and after the interview to assess the gain in information due to the interview. The draft write-up of each study was checked with each respondent for accuracy and changed if necessary. RESULTS: The literature review identified a highly diverse literature but confirmed that the 'payback' framework pioneered by Buxton and Hanney was the most widely used and most appropriate model available. The review also confirmed that impact on knowledge generation was more easily quantified than that on policy, behaviour or especially health gain. The review of the included studies indicated a higher level of impact on policy than is often assumed to occur. The survey showed that data pertinent to payback exist and can be collected. The completed questionnaires showed that the HTA Programme had considerable impact in terms of publications, dissemination, policy and behaviour. It also showed, as expected, that different parts of the Programme had different impacts. The Technology Assessment Reports (TARs) for the National Institute for Health and Clinical Excellence (NICE) had the clearest impact on policy in the form of NICE guidance. Mean publications per project were 2.93 (1.98 excluding the monographs), above the level reported for other programmes. The case studies revealed the large diversity in the levels and forms of impacts and the ways in which they arise. All the NICE TARs and more than half of the other case studies had some impact on policy making at the national level whether through NICE, the National Screening Committee, the National Service Frameworks, professional bodies or the Department of Health. This underlines the importance of having a customer or 'receptor' body. A few case studies had very considerable impact in terms of knowledge production and in informing national and international policies. In some of these the principal investigator had prior expertise and/or a research record in the topic. The case studies confirmed the questionnaire responses but also showed how some projects led to further research. CONCLUSIONS: This study concluded that the HTA Programme has had considerable impact in terms of knowledge generation and perceived impact on policy and to some extent on practice. This high impact may have resulted partly from the HTA Programme's objectives, in that topics tend to be of relevance to the NHS and have policy customers. The required use of scientific methods, notably systematic reviews and trials, coupled with strict peer reviewing, may have helped projects publish in high-quality peer-reviewed journals. Further research should cover more detailed, comprehensive case studies, as well as enhancement of the 'payback framework'. A project that collated health research impact studies in an ongoing manner and analysed them in a consistent fashion would also be valuable.

Elevated platelet beta-secretase activity in mild cognitive impairment.

BACKGROUND/AIMS: We have recently reported that platelet activity of the rate-limiting enzyme for beta-amyloid peptide production is elevated in established Alzheimer's disease. Laboratory investigation of the very early stages of dementia provides an opportunity to investigate pathological mechanisms before advanced disease hinders interpretation. Mild cognitive impairment (MCI) exists prior to obvious dementia, and is associated with increased risk of conversion to overt disease. METHODS: We developed and used a fluorimetric assay to quantify platelet membrane beta-secretase activity in 52 patients with MCI and 75 controls. RESULTS: Platelet membrane beta-secretase activity was 24% higher in individuals with MCI compared to controls (p = 0.001, unpaired t test with Welch correction). CONCLUSION: Elevated platelet beta-secretase activity in subjects with MCI is an area for further study in relation to the etiology and diagnosis of MCI.

Expression and activity of beta-site amyloid precursor protein cleaving enzyme in Alzheimer's disease.

Several lines of evidence indicate that the Abeta peptide is involved at some level in the pathological process that results in the clinical symptoms of AD (Alzheimer's disease). The N-terminus of Abeta is generated by cleavage of the Met-Asp bond at position 671-672 of APP (amyloid precursor protein), catalysed by a proteolytic activity called beta-secretase. Two 'beta-secretase' proteases have been identified: BACE (beta-site APP-cleaving enzyme) and BACE2. The cause of sporadic AD is currently unknown, but some studies have reported elevated BACE/beta-secretase activity in brain regions affected by the disease. We have demonstrated that robust beta-secretase activity is also detectable in platelets that contain APP and release Abeta. This review considers the current evidence for alterations in beta-secretase activity, and/or alterations in BACE expression, in post-mortem brain tissue and platelets from individuals with AD.

The balance between isoforms of the prickle LIM domain protein is critical for planar polarity in Drosophila imaginal discs.

The tissue polarity mutants in Drosophila include a set of conserved gene products that appear to be involved in the control of cytoskeletal architecture. Here we show that the tissue polarity gene prickle (pk) encodes a protein with a triple LIM domain and a novel domain that is present in human, murine, and Caenorhabditis elegans homologs which we designate PET. Three transcripts have been identified, pk, pkM, and sple, encoding 93-, 100-, and 129-kD conceptual proteins, respectively. The three transcripts span 70 kb and share 6 exons that contain the conserved domains. The pk and sple transcripts are expressed with similar tissue-specific patterns but have qualitatively different activities. The phenotypes of pk mutants, and transgenic flies in which the different isoforms are overexpressed show that the balance between Pk and Sple is critical for the specification of planar polarity. In addition, these phenotypes suggest a tessellation model in which the alignment of wing hairs is dependent on cell shape and need not reflect fine-grained positional information. Lack of both pk and sple transcripts gives a phenotype affecting the whole body surface that is similar to those of dishevelled and frizzled (fz) suggesting a functional relationship between pk and fz signaling.

Topological constraints on transvection between white genes within the transposing element TE35B in Drosophila melanogaster.

The transposable element TE35B carries two copies of the white (w) gene at 35B1.2 on the second chromosome. These w genes are suppressed in zeste-1 (z1) mutant background in a synapsis-dependent manner. Single-copy derivatives of the original TE35B stock give red eyes when heterozygous, but zeste eyes when homozygous. TE35B derivatives carrying single, double or triple copies of w were crossed to generate flies carrying from two to five ectopic w genes. Within this range, z1-mediated suppression is insensitive to copynumber and does not distinguish between w genes that are in cis or in trans. Suppression does not require the juxtaposition of even numbers of w genes, but is extremely sensitive to chromosomal topology. When arranged in a tight cluster, in triple-copy TE derivatives, w genes are nonsuppressible. Breakpoints falling within TE35B and separating two functional w genes act as partial suppressors of z1. Similarly, breakpoints immediately proximal or distal to both w genes give partial suppression. This transvection-dependent downregulation of w genes may result from mis-activation of the X-chromosome dosage compensation mechanism.

Genetic and cytogenetic analysis of the 43A-E region containing the segment polarity gene costa and the cellular polarity genes prickle and spiny-legs in Drosophila melanogaster.

A cytogenetic analysis of the 43A-E region of chromosome 2 in Drosophila melanogaster is presented. Within this interval 27 complementation groups have been identified by extensive F2 screens and ordered by deletion mapping. The region includes the cellular polarity genes prickle and spiny-legs, the segmentation genes costa and torso, the morphogenetic locus sine oculis and is bounded on its distal side by the eye-color gene cinnabar. In addition 19 novel lethal complementation groups and two semi-lethal complementation groups with morphogenetic escaper phenotypes are described.

Polytene chromosome microdissection and molecular genome mapping in Drosophila and other dipterans.

We are constructing a molecular physical map of the Drosophila melanogaster genome, using microdissection of polytene chromosomes as a source of region specific probes applied to direct mapping work. Chromosome microdissection has been used for a low resolution map of the Anopheles gambiae s.s. genome.