Neurobiological studies of reading and reading disability.

UNLABELLED: Evidence from neuroimaging studies, including our own, suggest that skilled word identification in reading is related to the functional integrity of two consolidated left hemisphere (LH) posterior systems: a dorsal (temporo-parietal) circuit and a ventral (occipito-temporal) circuit. This posterior system appears to be functionally disrupted in developmental dyslexia. Relative to nonimpaired readers, reading-disabled individuals demonstrate heightened reliance on both inferior frontal and right hemisphere posterior regions, presumably in compensation for the LH posterior difficulties. We propose a neurobiological account suggesting that for normally developing readers, the dorsal circuit predominates at first, and in conjunction with premotor systems, is associated with analytic processing necessary for learning to integrate orthographic with phonological and lexical semantic features of printed words. The ventral circuit constitutes a fast, late-developing, word form system, which underlies fluency in word recognition. LEARNING OUTCOMES: As a result of this activity, (1) the participant will learn about a model of lexical processing involving specific cortical regions. (2) The participant will learn about evidence which supports the theory that two dorsal LH systems may be disrupted in developmental dyslexia. (3) The participant will learn that individuals with reading impairment may rely on other regions of the brain to compensate for the disruption of posterior function.

Aberrant expression of G(1)/S regulators is a frequent event in sporadic pituitary adenomas.

Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution we have simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.

A year's experience of the Roche Advantage II glucose test strip.

The findings of a ward-based quality assurance scheme for near-patient blood glucose measurement have been analysed statistically. They provide evidence to support an improved analytical performance of a new glucose meter test strip, the Advantage II test strip, which has been in use in our hospital for over a year.

Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-beta1-induced expression of p15/INK4B.

Transforming growth factor beta (TGF-beta) is a member of a family of cytokines that regulate differentiation and proliferation in a wide variety of tissues including the pituitary gland. In both the normal pituitary and tumorous cell lines TGF-beta1 has anti-proliferative activity, however the intracellular mechanisms responsible have not been defined. In the pituitary derived cell line GH(3), p27(Kip1), a key regulator of G(1)/S transition is not expressed, suggesting that this protein is not an effector of the anti-proliferative response following TGF-beta1 treatment. Among other TGF-beta responsive cell cycle regulators p15(Ink4b) has been shown to have anti-proliferative effects associated with cell cycle arrest in other cell types. We therefore examined p15(Ink4b) expression in response to TGFbeta-1 to determine if this cyclin dependent kinase inhibitor was responsible for anti-proliferative activity in GH(3) cells. Treatment of GH(3) cells with TGF-beta1 (0.5-30 ng/ml) showed significant dose dependent growth inhibition (P<0.001) as assessed by viable cell counts. Maximum growth inhibition (66%) was observed following treatment with 2 ng/ml TGF-beta1. FACS analysis carried out in parallel with the growth studies showed treatment was associated with a decrease in the proportion of cells in S-phase (22-9%) and a significant increase in the G(1) fraction from 58 to 75% relative to controls (P<0.001). The absence of a sub G(1) fraction and reversibility of the G(1) arrest over three cycles showed that these changes were not due to either an apoptotic response or cytoxicity, respectively. Semi-quantitative RT-PCR and Western blot analysis showed no change in the expression level of cyclin dependent kinase 4 (CDK4), p16(Ink4a) or p21(Cip1). However, p15(Ink4b) mRNA and protein levels showed a 10 and 8 -fold induction, respectively. Increased levels of p15(Ink4b) were accompanied by a shift in the phosphorylation status of pRb toward its active hypophosphorylated form. Furthermore, studies of the kinetics of p15(Ink4b) induction showed that arrest of cells in G(1) is preceded by induction of p15(Ink4b) mRNA and protein. These investigations would suggest that p15(Ink4b) is a functional effector of TGF-beta1 mediated cell cycle arrest in GH(3) cells. However, our present studies cannot determine if it is the sole mediator. Identification of intracellular target(s) that mediate responses to anti-proliferative signals will increase our understanding of these pathways and aberrations responsible for their dysfunction in tumorigenesis.

Functional neuroimaging studies of reading and reading disability (developmental dyslexia).

Converging evidence from a number of neuroimaging studies, including our own, suggest that fluent word identification in reading is related to the functional integrity of two consolidated left hemisphere (LH) posterior systems: a dorsal (temporo-parietal) circuit and a ventral (occipito-temporal) circuit. This posterior system is functionally disrupted in developmental dyslexia. Reading disabled readers, relative to nonimpaired readers, demonstrate heightened reliance on both inferior frontal and right hemisphere posterior regions, presumably in compensation for the LH posterior difficulties. We propose a neurobiological account suggesting that for normally developing readers the dorsal circuit predominates at first, and is associated with analytic processing necessary for learning to integrate orthographic features with phonological and lexical-semantic features of printed words. The ventral circuit constitutes a fast, late-developing, word identification system which underlies fluent word recognition in skilled readers.

The cerebellum's role in reading: a functional MR imaging study.

BACKGROUND AND PURPOSE: Long considered to have a role limited largely to motor-related functions, the cerebellum has recently been implicated as being involved in both perceptual and cognitive processes. Our purpose was to determine whether cerebellar activation occurs during cognitive tasks that differentially engage the component processes of word identification in reading. METHODS: Forty-two neurologically normal adults underwent functional MR imaging of the cerebellum with a gradient-echo echo-planar technique while performing tasks designed to study the cognitive processing used in reading. A standard levels-of-processing paradigm was used. Participants were asked to determine whether pairs of words were written in the same case (orthographic processing), whether pairs of words and non-words rhymed with each other, respectively (phonologic assembly), and whether pairs of words belonged to the same category (semantic processing). Composite maps were generated from a general linear model based on a randomization of statistical parametric maps. RESULTS: During phonologic assembly, cerebellar activation was observed in the middle and posterior aspects of the posterior superior fissure and adjacent simple lobule and semilunar lobule bilaterally and in posterior aspects of the simple lobule, superior semilunar lobule, and inferior semilunar lobule bilaterally. Semantic processing, however, resulted in activation in the deep nuclear region on the right and in the inferior vermis, in addition to posterior areas active in phonologic assembly, including the simple, superior semilunar, and inferior semilunar lobules. CONCLUSION: The cerebellum is engaged during reading and differentially activates in response to phonologic and semantic tasks. These results indicate that the cerebellum contributes to the cognitive processes integral to reading.

Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G1 arrest in the AtT20 pituitary tumor cell line.

Recent studies have shown that methylation of the CpG island within the p16/CDKN2A gene is associated with an absence of p16 protein in human pituitary tumors. However, the effect of restoration of p16 protein expression in this tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system. Initial experiments showed that the p16/CDKN2A gene was not expressed, whereas a transcript for RB1 was detected as assessed by RT-PCR. Further studies showed the p16/CDKN2A gene to be homozygously deleted. The absence of p16/CDKN2A and presence of RB1, the down-stream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model system. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activated cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G1 arrest (80-82%). Western blot analysis showed concomitant expression of p16 protein in arrested cells and a shift in the phosphorylation status of pRB toward its hypophosphorylated form. To further confirm that expression of p16/CDKN2A mimicked its in vivo role, reversibility was assessed using alternate cycles in the presence and absence of inducer (isopropyl-1-thio-beta-D-galactopyranoside). Over three cycles the absence of induced expression of p16/CDKN2A resulted in release from G1 arrest. These results show that, in a pituitary cell line model, restoration of p16 expression is indeed sufficient to arrest cells in G1 and inhibit cell proliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.

Identity priming in English is compromised by phonological ambiguity.

If it takes longer to achieve a single phonological representation for inconsistent words (e.g., BOWL) than for consistent words (e.g., BENT), and if phonological coherence is pivotal to visual word recognition, then identity priming should depend on consistency. This hypothesis was evaluated in naming and lexical decision within a 4-field presentation sequence of mask-prime-mask-target. The prime-target stimulus onset asynchrony (SOA) was either 114 or 244 ms (with prime durations, respectively, of 43 and 129 ms). Four experiments compared identity primes such as BOWL and BENT, which were equated, on average, for total number of friendly and unfriendly neighbors, bigram frequency, and number of 1-letter-different neighbors. In both tasks, BENT primed itself better than BOWL primed itself, with the difference being larger at the shorter SOA. Word processing is constrained primarily by the rate of achieving a coherent phonological code.

Methylation mechanisms in pituitary tumorigenesis.

Methylation is essential for embryonic development, however aberrant methylation of CpG islands associated with the tumour suppressor genes (TSGs) and leading to gene silencing is found in numerous tumour types. The TSG p16/CDKN2A is involved in the genesis of many tumour types and frequent methylation of the CpG island of the p16/CDKN2A gene is associated with loss of protein expression in pituitary tumours. In addition, CpG sites are mutational hotspots and abnormal methylation patterns have been shown to lead to genetic instability, predisposing to, and preceding allelic loss. Although several studies of pituitary tumours have shown loss of genetic material at known and putative TSGs loci, studies of the retained alleles have revealed infrequent mutation. Equally, for several other TSGs no mechanisms have been described for their reduced expression. Methylation may represent a unifying theme, responsible in some cases for an absence or reduced expression and in other cases predisposing to allelic loss that may or may not encompass a TSG. In several tumour types treatment of tumours or their cognate cell lines with demethylating agents induces expression of previously methylated genes. Using the mouse corticotroph cell line AtT20 as a model system, transfection studies showed restoration of growth control through induction of ectopically expressed p16/CDKN2A. These effects were reversed by prior in vitro methylation of the constructs' CpG sites within the coding region of this gene. Methylation of an otherwise unmethylated CpG island renders a gene transcriptionally incompetent and clinically these genes represent attractive therapeutic targets since the gene is neither lost nor mutated, but may be reactivated. Future studies will no doubt describe more efficacious pharmacological interventions and identify the mechanisms responsible for the abnormal methylation patterns seen in tumours including those of pituitary origin.

Pain in the assessment of total knee replacement.

The results of total knee replacement (TKR) are commonly assessed by survival analysis using revision as the endpoint. We have used the assessment of pain by a patient-based questionnaire as an alternative. In one hospital, 1429 TKRs were inserted by 66 surgeons between 1987 and 1993. The survival at seven years, with revision as the endpoint, was 97.5% (CI 94 to 100). There were no significant differences between the three different types of implant used, the AGC, the IB2 and the Nuffield Knee. When the endpoint was the development of moderate pain, the survival at seven years for the AGC knee was 72% and that for the IB2 was similar. Significantly more patients (p = 0.007) with the Nuffield Knee, however, had developed moderate pain. Using revision as the endpoint, it is difficult to discriminate between the various types of TKR, but this can be achieved using pain. In this investigation 30% of the patients reported moderate pain at some stage by seven years from operation.

Urinary microalbumin measurement using a homogeneous liposomal immunoassay.

A homogeneous colorimetric immunoassay which has been developed for urinary microalbumin utilizes complement-mediated immunolysis of liposomes containing the dye, sulphorhodamine B. Unlike a previously described model complement-mediated liposomal assay for serum albumin (Frost et al., 1994) which was competitive, this assay uses a sandwich-type format and Fab' (antialbumin)-coated liposomes to increase the assay sensitivity. The liposomal assay, performed using a Cobas Bio analyser (Roche, Welwyn Garden City, UK), gave an acceptable correlation with a radioimmunoassay (NETRIA, London, UK): r = 0.94; y (liposomal assay) = 1.09 x (radioimmunoassay) - 1.54 mg/1. The imprecisions of the assays were similar and matrix effects due to the use of urine samples were determined to be acceptably small. The assay demonstrates the advantage of using Fab'-coated liposomes in sandwich-type liposomal immunoassays over liposomes coated with intact antibody, which failed to elicit complement-mediated immunolysis.

Novel homogeneous liposomal immunoassay for colorimetric estimation of serum IgG anticardiolipin antibodies.

Liposomes entrapping the dye sulforhodamine B were used to develop an assay for anticardiolipin antibodies (ACAs). In the presence of magnesium ions, IgG ACAs induced liposomal lysis and the resulting absorbance changes were dependent on the amount of ACAs present. The liposomal assay (y) showed similar intraassay imprecision and detection limit to an ELISA for IgG ACAs (x), with a correlation coefficient of 0.90 (y = 1.05x - 1.61). No correlation with ELISA IgM ACA measurements was observed. Although ELISA remains the method of choice, particularly in examining different ACA classes, the liposomal assay offers advantages of speed and potential for processing large numbers of samples for IgG ACAs. This may facilitate study of the significance of increased IgG ACAs in the groups of conditions with which they are associated, and perhaps enable more laboratories to perform the test.

Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells.

Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.

Rat hepatocyte hyaluronan/glycosaminoglycan binding proteins: evidence for distinct divalent cation-independent and divalent cation-dependent activities.

We have previously shown (Biochemistry, 29, 10425, 1990) that hepatocytes contain intracellular specific binding sites for hyaluronan (HA). Although HA-binding activity is not dependent on divalent cations, it is increased in the presence of Ca+2. Here we report that a novel photoaffinity HA derivative (ASD-HA) crosslinks specifically to different proteins in permeable cells in the presence or absence of Ca+2. With Ca+2 present, two proteins of approximately 24 kD and 43 kD were labeled. Additionally, a broad zone of specific crosslinking was observed in the region of 40-100 kD. However, in the presence of the chelator EGTA this zone was absent and the 24 and 43 kD proteins were also not cross-linked to the HA photoaffinity derivative. In the absence of Ca+2, only a 54 kD protein was specifically labeled. The results indicate that different intracellular hepatocyte proteins are responsible for the Ca+2-independent and the Ca+2-dependent binding of HA.

Atrial natriuretic peptide degradation by CPA47 cells: evidence for a divalent cation-independent cell-surface proteolytic activity.

Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88% of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41% of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

Identification of the Ca(2+)-independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linking reagent.

The Ca(2+)-independent endocytic hyaluronan (HA) receptor in rat liver sinusoidal endothelial cells (LECs) was identified using a novel cross-linking derivative of HA. The heterobifunctional, photoactivatable, reducible reagent sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) was coupled to the terminal amino group of uniquely modified HA-amine oligosaccharides (M(r) approximately 60,000) and subsequently iodinated. 125I-ASD-HA bound to cultured LECs with similar specificity and affinity as a previously characterized 125I-HA-amine/Bolton-Hunter adduct. Permeabilized LECs were incubated with 125I-ASD-HA with 10 mM EGTA and photolysed with UV light. Detergent extracts were reduced to release the HA oligosaccharides and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Two polypeptides were consistently and equally labeled at M(r) = 175,000 and 166,000. Photoaffinity labeling of these two proteins was virtually identical in cultured LECs or membranes and was competed greater than 90% with a 100-fold excess of HA. As with the previously characterized bona fide LEC HA receptor, cross-linking was also competed by chondroitin sulfate and heparin, but less efficiently by chondroitin and not with galacturonan. We conclude that the Ca(2+)-independent LEC HA receptor is composed of at least two polypeptides of M(r) approximately 175,000 and 166,000 and may exist as a heterodimer of M(r) approximately 340,000. We also conclude that the LEC HA receptor is distinct from the CD44 family of HA-binding proteins.

Antibody-coated liposomes as a particulate solid phase for immunoassays. Measurement of urinary 'micro-albumin'.

A novel use of liposomes as a solid phase material achieving separation in immunoassays is described. Antibody-coated liposomes were prepared and used as a particulate solid phase in a radioimmunoassay procedure for urinary albumin. The assay was compared to a liquid phase albumin radioimmunoassay. The potential benefits of liposomes over other particulate solid phases are discussed. The use of liposomes in this manner need not be restricted to radioimmunoassay but should also be applicable to other immunoassays using alternative non-isotopic labels.

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)