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Biomedical subjects

M Dennis

Publications and source records attributed to M Dennis.

At least 91 records · Page 5Linked to original sources

Pharmacological and molecular characterization of the neurotensin receptor expressed in Sf9 cells.

The rat neurotensin receptor was expressed in Spodoptera frugiperda insect (Sf9) cells using infection with a recombinant baculovirus. Immunoblot experiments performed with an antibody raised against the C-terminus of the receptor showed major bands at 47 (corresponding to the unglycosylated receptor protein) and 50 kDa, and minor bands at 65 and 36 kDa. The expressed receptor bound 125I-neurotensin with high affinity, was coupled to endogenous G-proteins, and agonist-induced inositol phosphate production was observed at early times after infection. These results show that the rat neurotensin receptor retains functional properties when expressed in the heterologous insect cell system.

Animals↗

The effects of knowledge availability and knowledge accessibility on coherence and elaborative inferencing in children from six to fifteen years of age.

Two experiments are presented in which a novel knowledge base was acquired by 6- to 15-year-old children prior to hearing a multiepisode story, and where inferences from the story drew only on that knowledge base. Making knowledge equally available to all children did not attenuate age-related differences in either coherence or elaborative inferencing. Easily accessible knowledge was generally twice as likely to be used to make inferences during text comprehension as was knowledge that took longer to retrieve, though knowledge accessibility was more important for coherence inferencing in younger than in older children. Children made more coherence than elaborative inferences in the context of text comprehension, even though elaborative inferencing was more frequent in a simpler processing situation. Within the context of an available knowledge base, the results provide evidence for the importance of knowledge accessibility in children's inferencing, and for the changing developmental relevance of knowledge accessibility for coherence and elaborative inferencing.

Adolescent↗

Neuropsychological sequelae of the treatment of children with medulloblastoma.

When a malignant tumor invades the child's cerebellum, the cost of successful treatment is often significant cognitive morbidity. A review of neuropsychological outcome revealed that survivors of childhood medulloblastoma (MB) have long-term deficits in intelligence, memory, language, attention, academic skills, psychosocial function, and a compromised quality of life. These deficits varied with chronological age at tumor diagnosis and/or adjuvant treatment, type and duration of presenting symptoms, tumor extension beyond the cerebellum, a history of adjuvant radiation treatment, and time since treatment. The effects on neuropsychological outcome of other factors, such as post-surgical hydrocephalus, were less clear. To understand the interaction between two factors predictive of outcome, age at diagnosis and time since treatment, we analyzed IQ results for a new sample of 25 surgically-treated and radiated MB survivors, and found that age at diagnosis and time since treatment made separable contributions to intellectual morbidity. PIQ appeared to measure some general effects of diffuse cerebral insult because it varied with chronological age of the child at tumor diagnosis but was relatively constant in magnitude, once established. VIQ, in contrast, was somewhat less sensitive to age at diagnosis in treated MB survivors, but declined with time since treatment. These results are important for understanding the academic attainments and continuing rehabilitation needs of childhood MB survivors, because they suggest that these children progressively fail to assimilate new verbally-based knowledge at a developmentally-appropriate rate.

Age Factors↗

Interaction between growth factors and kinins in arterial smooth muscle cells.

Bradykinin receptors are present on vascular smooth muscle cells; however, the regulation and biological function of these receptors is unclear. To address these questions the interaction between growth factors and kinins in cultured arterial smooth muscle cells has been examined. Based upon the data a hypothesis is presented that platelet-derived growth factor (PDGF) upregulates cell surface bradykinin B2 receptors on arterial smooth muscle cells. The biological effect of the increase in B2 receptors is currently unclear but under certain conditions they may enhance mitogenesis. These mitogenic effects however, are strongly opposed by the effects of bradykinin acting via a B1-type of receptor which mediates potent inhibition of growth factor-induced mitogenesis.

Animals↗

Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques.

Prevention of sexually transmitted HIV infection was investigated in macaques by immunization with a recombinant SIV (simian immunodeficiency virus) envelope gp 120 and core p27 vaccine. In two independent series of experiments, we used the novel targeted iliac lymph node (TILN) route of immunization, aiming close to the iliac lymph nodes draining the genitorectal mucosa. Rectal challenge with the SIVmac 32H J5 molecular clone in two series induced total protection in four out of seven macaques immunized by TILN, compared with infection in 13 of 14 unimmunized macaques or immunized by other routes (P = 0.025). The remaining three macaques showed either a decrease in viral load ( > 90%) or transient viremia, indicating that all seven TILN-immunized macaques showed total or partial protection (P = 0.001). Protection was associated with significant increase in the iliac lymph nodes of IgA antibody-secreting cells to p27 (P < 0.02), CD8-suppressor factor (P < 0.01), and the chemokines RANTES and MIP-1 beta (P < 0.01).

Animals↗

Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain.

In this work, the molecular forms of the rat neurotensin receptor (NTR) expressed in transfected Chinese hamster ovary (CHO) cells, in infected Sf9 insect cells and in rat cerebral cortex were immunologically detected by means of an anti-peptide antibody raised against a fragment of the third intracellular loop of the receptor. Immunoblot experiments against a fusion protein indicated that the anti-peptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence within the NTR. In immunoblot analysis of membranes from NTR-transfected CHO cells, high levels of immunoreactivity were observed between 60 and 72 kDa, while only a faint labelling was observed at 47 kDa, the molecular mass deduced for the rat NTR cDNA. The bands of high molecular mass were no longer observed after deglycosylation of membrane proteins by peptide N-glycosidase F, indicating that they represented glycosylated forms of the receptor. Extracts of membranes derived from baculovirus-infected Sf9 insect-cells expressing the NTR provided a quite different immunoblot pattern, since the major band detected in that case was at 47 kDa, the molecular size of the non-glycosylated receptor. Taken together, these data show that, while most of the NTR protein was glycosylated in CHO cells, it was unglycosylated in Sf9 insect-cells. In addition, molecular sizes of the receptor proteins observed in these two cell lines differed from those obtained for the NTR endogenously expressed in the rat cerebral cortex of 7 day-old rats, where bands at 56 and 54 kDa were detected. Binding experiments carried out on membrane preparations obtained from baculovirus-infected Sf9 cells demonstrated that the immunogenic sequence was still accessible to the antibody when the receptor was embedded in the cell membrane. Immunohistochemical studies carried out on both transfected CHO cells and infected Sf9 cells confirmed this interpretation and further indicated that the antibody could be applied in the visualization of the receptor.

Amino Acid Sequence↗

Early suppression of SIV replication by CD8+ nef-specific cytotoxic T cells in vaccinated macaques.

In order to develop a successful subunit vaccine against infection with the human immunodeficiency virus (HIV), protective immune effector functions must be identified. Until now, there has been only indirect evidence that HIV-specific cytotoxic T lymphocytes (CTLs) fulfill this role. Using the macaque simian immunodeficiency virus (SIV) model, the protective potential of nef-specific CTLs, stimulated by vaccination, was examined in animals challenged with a high intravenous dose of the pathogenic simian immunodeficiency virus, SIVmac251(32H)(pJ5). An inverse correlation was found between the vaccine-induced nef-specific CTL precursor frequency and virus load measured after challenge. In addition, the early decline in viraemia, observed in both vaccinated and unvaccinated control animals was associated with the development of virus-specific CTL activity and not with the presence of virus-specific neutralizing antibodies. The results imply that vaccines that stimulate strong CTL responses could protect against HIV infection.

Animals↗

Patients' and carers' satisfaction with acute stroke management.

We set out to discover how satisfied patients and carers were with existing stroke services. We prospectively identified 164 consecutive patients admitted to a department of general medicine with acute stroke and collected data using a satisfaction questionnaire in a semi-structured interview. of the 110 (67%) survivors, 65 (59%) patients and 80 (73%) carers completed an interview. We also interviewed 34 bereaved carers. Most patients (97%), carers (92%) and bereaved carers (94%) were satisfied with care overall. However, 30 (46%) of our patients, 53 (66%) carers and 18 (53%) bereaved carers who were satisfied with care overall expressed dissatisfaction with at least one component. Carers of patients were significantly more dissatisfied than the patients themselves with the amount of information given and with social work intervention. Carers of patients who were unable to respond to the questionnaire themselves, usually because of cognitive difficulties, were the most dissatisfied group even when compared with bereaved carers. Measuring satisfaction can alert one to deficiencies within a service, aiding service development, but interpreting patients' and carers' overall satisfaction. Efforts to improve communication, discharge planning and follow-up, aimed at carers as well as the patients themselves, are likely to be rewarded with improved satisfaction with stroke services.

Acute Disease↗

Hospital services for patients with acute stroke in the United Kingdom: the Stroke Association Survey of consultant opinion.

A national postal survey of all UK hospital consultant geriatricians, general physicians and neurologists was performed in 1992/3 in order to describe the provision of hospital stroke services in the United Kingdom and to assess whether the recommendations of the King's Fund consensus conference on stroke had been widely implemented. Of 3478 survey forms, 2923 (84%) were returned and, of these 1953 (67%) consultants indicated that they routinely cared for patients with acute stroke. On their stated estimates, the survey respondents had admitted approximately 107,000 patients with acute stroke in the previous year, 40% of whom were cared for by geriatricians. Sixty-three per cent (1239/1953) worked in District General Hospitals. Few (5%) had access to an acute stroke unit, and a majority (51%) of consultants were uncertain of the benefits of such units. Less than half (44%) had access to a specialized stroke rehabilitation unit, but a majority (68%) were certain of the benefits of stroke rehabilitation units. Although a majority of consultants had on-site CT scanning, about a third of all UK stroke patients were admitted to a hospital without on-site CT facilities. Most (90%) consultants would want a CT scan themselves if they had a stroke. Only about a third of consultants were aware of a recent audit of stroke care in their hospital, or had a hospital policy for the implementation of minimum standards of stroke care, and less than half routinely provided written information for patients or carers. This survey illustrated that five years after the publication of the King's Fund consensus statement on the treatment of patients with acute stroke UK hospital stroke services are still poorly organized. Access to CT scanning for stroke patients is improving, but is still insufficient.

Acute Disease↗

Serotonergic antagonists differentially inhibit spontaneous activity and decrease ligand binding capacity of the rat 5-hydroxytryptamine type 2C receptor in Sf9 cells.

The activities of serotonergic antagonists as inverse agonists at the rat 5-hydroxytryptamine (5-HT)2C serotonin receptor were compared with their potencies in promoting receptor "down-regulation," after expression of the recombinant receptor in the baculovirus/Sf9 insect cell system. Baculovirus expression yielded high levels of 5-HT2C receptors (up to 10(6) receptors/cell), which were functionally coupled to polyphosphoinositide turnover in Sf9 cells through a pertussis toxin-insensitive pathway. The expressed receptor exhibited spontaneous activation of inositol phosphate production, which was inhibited in a dose-dependent manner by serotonergic antagonists, consistent with inverse agonist activity. The potencies of antagonists as inverse agonists correlated with their respective binding affinities determined in competition binding studies with membrane preparations. The maximal inhibition of spontaneous activity ranged from 32% inhibition for mianserin to no effect for spiroxatrine, indicating that antagonists differ in their intrinsic inverse efficacies. Antagonist treatment of intact Sf9 cells or membranes containing the 5-HT2C receptor, followed by washout of residual drug, resulted in a decrease (up to 90%) in the number of binding sites for [3H]mesulergine and [3H]5-HT, with no change in the affinity for [3H]mesulergine. The decrease in binding was irreversible, was not due to the presence of residual antagonist, and was not observed after treatment with agonists. This effect of antagonists in membranes was dose dependent, but the rank order of potency was clearly different from that for inverse agonist activity, indicating that the two effects reflect distinct actions of antagonists at the 5-HT2C receptor. The relative abilities of antagonists to produce loss of binding showed a good correlation with their reported abilities to down-regulate 5-HT2 receptors in vivo after chronic treatment, suggesting that these actions reflect the same underlying process.

Animals↗

Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain.

Polypeptide hormones and growth factors have long been known to internalize into peripheral target cells as a result of their interaction with cell surface receptors. Studies in culture have suggested that certain neuropeptides might undergo a similar type of translocation in neurons. To investigate this possibility in adult mammalian brain, we have examined by confocal laser microscopy the events that follow the binding of fluorescein-tagged derivatives of the tridecapeptide neurotensin to basal forebrain cholinergic cells. Our results demonstrate a selective time- and temperature-dependent internalization of fluo-neurotensin in these cells. This internalization is receptor mediated, proceeds from the entire somatodendritic membrane of the cells, and utilizes endosome-like organelles which are mobilized from dendrites to perikarya and from the periphery of the cell to its perinuclear region. Parallel studies carried out on Sf9 insect cells expressing the rat neurotensin receptor from a recombinant baculovirus indicated that the internalization process involves receptor-ligand complexes and not merely the fluorescent peptide itself. These data suggest that receptor internalization plays a role in neuropeptide signaling in the brain and that it can be harnessed for selective identification of neuropeptide target cells.

Animals↗

Seroprevalence of Helicobacter pylori in residents of a hospital for people with severe learning difficulties.

OBJECTIVE: To assess the seroprevalence of Helicobacter pylori among the residents of a hospital for people with severe learning difficulties. DESIGN: Retrospective analysis of stored sera from the hospital residents and control sera from the local, non-residential population. METHODS: H. pylori immunoglobulin G antibody was measured in 424 hospital residents using an enzyme-linked immunosorbent assay, taking an antibody level of > 10 units/ml as evidence of H. pylori infection. The results were compared with 267 age- and sex-matched controls. RESULTS: Seropositivity rates were significantly higher in the hospital residents than in controls for all ages. This was most marked among those under 40 years of age (87 versus 24% H. pylori-positive for residents and controls, respectively; P < 0.001). The overall seropositivity rates were 87 and 43% for residents and controls, respectively (P < 0.001). CONCLUSIONS: This English study of H. pylori seroprevalence in an institutionalized population is the largest to date and confirms the very high seropositivity rates found by previous studies in Australia. Our findings may have significance for the future health of these patients and for the possible modes of transmission of H. pylori.

Adolescent↗

Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor.

The regulation and post-translational modifications of the human dopamine D1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D1 receptor or a DNA encoding a c-myc epitope tagged dopamine D1 receptor (c-myc-dopamine D1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D1 and c-myc-dopamine D1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D1 receptor, the photoaffinity label [125I](3-methyl-2-[4'-azidophenyl]-2,3,5-tetrahydro-2H-3-benzazepine) ([125I]MAB) bound specifically upon photolysis. A major broad band of approximately 48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

The content of narrative discourse in children and adolescents after early-onset hydrocephalus and in normally developing age peers.

The development of narrative content was studied in 100 children aged 6-15 years (49 with early-onset hydrocephalus and 51 age-matched controls) by analyzing transcripts of oral texts produced from their narrations of two fairy tales. In relation to those of their age-matched peers, the narratives of the children with hydrocephalus were less cohesive and less coherent. They conveyed less of the content needed for the narrative message, included more referentially ambiguous material, included uninterpretable or implausible content, and were more verbose and less economic in quality. In relation to their age-matched peers, then, children with hydrocephalus produce narratives that are difficult to process, unclear, uneconomic, and less fully elaborated for meaning. These data add to an emerging body of information that shows children and adolescents with early-onset hydrocephalus to be at risk for several types of discourse and pragmatic impairments. The language of children with hydrocephalus is discussed with reference to the theoretical distinction between interpersonal pragmatic conventions and constraints relating to textual rhetoric (processability, clarity, economy, and expressivity). By showing impaired textual rhetoric coexisting with apparently preserved interpersonal rhetoric in individuals with developmental anomalies of brain development, the present data provide some support for a functional dissociation between the two classes of pragmatic constraints.

Adolescent↗

Phosphorylation and palmitoylation of the human D2L dopamine receptor in Sf9 cells.

We have expressed and biochemically characterized the human D2long (D2L) dopamine receptor isoform using the baculovirus/Sf9 cell system. The expressed receptor bound ligands with a pharmacological profile similar to that reported for neuronal and cloned D2L receptors expressed in mammalian cell lines. Dopamine binding to D2L receptor was sensitive to guanine nucleotides, indicating receptor coupling to endogenous G proteins. A D2L receptor-specific antibody identified two major protein species at approximately 44 kDa and at approximately 93 kDa in immunoblots, suggesting the presence of D2L receptor monomers and dimers. Both species were purified by immunoprecipitation from digitonin-solubilized preparation of cells expressing D2L receptor prelabeled with 32P(i) or [3H]-palmitate. These results constitute the first direct evidence for D2L receptor phosphorylation and palmitoylation.

Animals↗

Development and reliability of the Royal College of Physicians stroke audit form. UK Stroke Audit Group.

This article reports the development of an audit form for assessing the quality of stroke care from medical records. A consensus conference to define standards was followed by testing of pilot versions of the audit form. Then formal assessment of inter-rater reliability was carried out in four centres using 76 medical records, each assessed by two of eight auditors. Inter-rater reliability was acceptable, especially when medically qualified auditors were used.

Aged↗