Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans.

Transgenic Caenorhabditis elegans nematodes have been engineered to express potentially amyloidic human proteins. These animals contain constructs in which the muscle-specific unc-54 promoter/enhancer of C. elegans drives the expression of the appropriate coding regions derived from human cDNA clones. Animals containing constructs expressing the 42-amino acid beta-amyloid peptide (derived from human amyloid precursor protein cDNA) produce muscle-specific deposits immunoreactive with anti-beta-amyloid polyclonal and monoclonal antibodies. A subset of these deposits also bind the amyloid-specific dye thioflavin S, indicating that these deposits have the tinctural characteristics of classic amyloid. Co-expression of beta-peptide and transthyretin, a protein implicated in preventing the formation of insoluble beta-amyloid, leads to a dramatic reduction in the number of dye-reactive deposits. These results suggest that this invertebrate model may be useful for in vivo investigation of factors that modulate amyloid formation.

Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.

Age-related learning deficits in transgenic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta-APP), from which the beta-A4 peptide is derived, is considered to be central to the pathogenesis of Alzheimer disease (AD). Transgenic mice expressing the 751-amino acid isoform of human beta-APP (beta-APP751) have been shown to develop early AD-like histopathology with diffuse deposits of beta-A4 and aberrant tau protein expression in the brain, particularly in the hippocampus, cortex, and amygdala. We now report that beta-APP751 transgenic mice exhibit age-dependent deficits in spatial learning in a water-maze task and in spontaneous alternation in a Y maze. These deficits were mild or absent in 6-month-old transgenic mice but were severe in 12-month-old transgenic mice compared to age-matched wild-type control mice. No other behavioral abnormalities were observed. These mice therefore model the progressive learning and memory impairment that is a cardinal feature of AD. These results provide evidence for a relationship between abnormal expression of beta-APP and cognitive impairments.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats.

The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding beta-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G beta-homologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the beta-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of beta-transducin homologs. Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of beta-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Growth, survival, gene expression, microbiota and tryptic activity during feeding of Scophthalmus maximus first feeding larvae with beta-glucan (MacroGard)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

A randomized controlled trial to assess sunscreen application and beta carotene supplementation in the prevention of solar keratoses

Background: Solar keratoses (SKs) are among the strongest determinants of skin cancer, but little is known about the success of measures to control these common skin tumors. Objective: To determine whether daily sunscreen application and/or beta carotene supplementation retards the rate of occurrence of SKs in adults in the medium term. Design: Randomized controlled trial conducted between February 1992 and August 1996. Setting: General community of the subtropical township of Nambour, Australia (latitude, 26degrees south). Participants: A total of 1621 adults aged 25 to 74 years. Interventions: Participants were randomized to daily use of sunscreen (application of a high-protection sunscreen to their head, neck, arms, and hands every morning) or application of sunscreen at their usual discretionary rate. They were also randomly assigned to take either one 30-mg tablet of beta carotene or one placebo tablet each day. Main Outcome Measure: Change in the prevalent number of SKs in the intervention group relative to change in the control group. Results: The ratio of SK counts in 1994 relative to 1992 was lower in people randomized to daily sunscreen use (1.20; 95% confidence interval, 1.04-1.39) than in those randomized to discretionary sunscreen use (1.57; 95% confidence interval, 1.35-1.84). This 24% reduction is equivalent to the prevention of an average of I additional SK per person over that time. A reduction in the rate of change of SK prevalence was also seen in the sunscreen intervention group relative to the discretionary sunscreen group between 1994 and 1996, but it was not significant. No effect on the rate of change of prevalent SK counts was seen among those taking beta carotene supplements relative. to those taking placebo tablets. Conclusions: Daily application of sunscreen retarded the rate of SK acquisition among adults in a subtropical environment, while a beta carotene supplementation of 30 mg/d had no influence on the occurrence of SKs.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Extrusion of mixtures of starch and D-limonene encapsulated with beta-cyclodextrin: Flavour retention and physical properties

d-Limonene was encapsulated with beta-cyclodextrin to improve its retention during pre-added flavour starch extrusion. The objective of this work was to determine the effect of processing condition on the flavour retention and extrudate properties. Corn starch containing five levels of beta-cyclodextrin-d-limonene capsules (0-5%) were extruded at five different maximum barrel temperatures (133-167 degrees C) and screw speeds (158-242 rpm) using a twin screw extruder. The effect of these parameters on the flavour retention, expansion, texture, colour difference (Delta E), Water Absorption Index, Water Solubility Index, and residence time distribution (RTD) were investigated. Barrel temperature and capsule level predominantly influenced flavour retention and extrudate properties, while screw speed primarily affected extruder performances such as torque, die pressure, specific mechanical energy and RTD. (c) 2005 Elsevier Ltd. All rights reserved.

The distribution of senile plaques, neurofibrillary tangles and beta/A4 deposits in the hippocampus in Alzheimer's disease

The density of senile plaques (SP) and neurofibrillary tangles (NFT) was studied in Glees and Marsland stained sections of the hippocampus and parahippocampal gyrus (PHG) in 20 pateints with Alzheimer's disease. In addition, in six of the patients, the density of beta/A4 protein deposits, as revealed by immunohistochemistry and neurofibrillary changes demonstrated with the Gallyas stain, were studied in adjacent sections. The density of Glees SP and beta/A4 deposits was significantly greater in area CA1 of the hippocampus and in the subiculum than in the PHG. Hence, neurofibrillary degeneration appears to be a more important lesion than beta/A4 deposition in the hippocampus compared with the PHG. In addition, the detailed distribution of the lesions in the hippocampus could be explained if beta/A4/SP and NFT occur on the axon terminals and in the cell bodies respectively of the same neurons.

Correlations between the morphology of diffuse and primitive beta-amyloid (A beta) deposits and the frequency of associated cells in Down's syndrome

Correlations between the morphology of beta-amyloid (A beta) deposits and the frequency with which they are associated with neurons and glial cells were studied in Down's syndrome. The diameter of diffuse deposits was positively correlated with the frequency of large (> 25 microns) neuronal cell bodies in the isocortex and with glial cells in the hippocampus. Diameters of primitive deposits were positively correlated with glial cells in the hippocampus and with glial cells and neurons in the isocortex. Staining intensity was positively correlated with glial cells especially in the hippocampus. The data suggest that: (i) diffuse deposits develop from neurons and primitive deposits from glia; (ii) the size of A beta deposits depends on the numbers of neurons and glia; (iii) glial cells are also involved in the conversion of A beta to amyloid; and (iv) the increased density of primitive deposits in the hippocampus is determined by the high density of glial cells.

The ratio of diffuse to mature beta/A4 deposits in Alzheimer's disease varies in cases with and without pronounced congophilic angiopathy

The density of diffuse, primitive, classic and compact beta/A4 deposits was estimated in the cortex and hippocampus in Alzheimer's disease (AD) cases with and without pronounced congophilic angiopathy (CA). The total density of beta/A4 deposits in a given brain region was similar in cases with and without CA. Significantly fewer diffuse deposits and more primitive/classic deposits were found in the cases with CA. The densities of the primitive, classic and compact deposits were positively correlated in the cases without CA. However, no correlations were observed between the density of the mature subtypes and the diffuse deposits in these cases. In the cases with CA, the density of the primitive deposits was positively correlated with the diffuse but not with the classic deposits. The data suggest that the mature beta/A4 deposits are derived from the diffuse deposits and that the presence of pronounced CA enhances their formation.

Dispersion of classic beta-amyloid deposits around blood vessels in Alzheimer’s disease

The spatial pattern of the classic (‘cored’) type of beta-amyloid (Abeta) deposit was studied in the upper laminae of the superior temporal gyrus in 9 cases of sporadic Alzheimer’s disease (SAD). Abeta stained tissue was counterstained with collagen IV to study the relationships between the spatial distribution of the classic deposits and the blood vessel profiles. Both the classic deposits and blood vessel profiles were distributed in clusters. In all cases, there was a spatial correlation between the clusters of the classic deposits and the larger diameter (>10 micron) blood vessel profiles and especially the vertically penetrating arterioles. In only 1 case, was there a significant spatial correlation between the clusters of the classic deposits and the smaller diameter (<10 micron) capillaries. In 9/11 cases, the clusters of the classic deposits were significantly larger than those of the clusters of the larger blood vessels. In addition, the density of the classic deposits declined as a negative exponential function with distance from the vertically penetrating arterioles. These results suggest that the classic Abeta deposits cluster around the larger blood vessels in the frontal cortex and that diffusion of proteins from these blood vessels could be involved in the pathogenesis of the classic deposits in SAD.

The density of mature beta/A4 deposits is enhanced in Alzheimer's disease cases with pronounced congophilic angiopathy

The density of diffuse, primitive, classic and compact beta/A4 deposits was estimated in the cortex and hippocampus in Alzheimer's disease (AD) cases with pronounced congophilic angiopathy (CA). The total density of beta/A4 deposits in a brain region was similar in cases with and without CA. Significantly fewer diffuse deposits and more primitive/classic deposits were found in the cases with CA. The density of the primitive, classic and compact deposits were positively correlated in the cases without CA. However, no correlations were observed between the density of the mature subtypes and the diffuse deposits in these cases. In cases with CA, the density of the primitive deposits was positively correlated with the diffuse deposits but not with the classic deposits. The data suggest that the mature beta/A4 deposits are derived from the diffuse deposits and that the presence of pronounced CA enhances their formation.