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Biomedical subjects

G Elliott

Publications and source records attributed to G Elliott.

At least 55 records · Page 3Linked to original sources
PubMed1997

Intercellular trafficking and protein delivery by a herpesvirus structural protein.

We show that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin. VP22 movement was observed both after delivery of DNA by transfection or microinjection and during virus infection. Moreover, we demonstrate that VP22 trafficking occurs via a nonclassical Golgi-independent mechanism. Sensitivity to cytochalasin D treatment suggests that VP22 utilizes a novel trafficking pathway that involves the actin cytoskeleton. In addition, we demonstrate intercellular transport of a VP22 fusion protein after endogenous synthesis or exogenous application, indicating that VP22 may have potential in the field of protein delivery.

Actin Cytoskeleton↗
PubMed1997

National Institute of Mental Health Collaborative Multimodal Treatment Study of Children with ADHD (the MTA). Design challenges and choices.

The Collaborative Multimodal Treatment Study of Children with Attention Deficit Hyperactivity Disorder (ADHD), the MTA, is the first child multisite cooperative agreement treatment study of children conducted by the National Institute of Mental Health, Rockville, Md. It examines the long-term effectiveness of medication vs behavioral treatment vs both for treatment of ADHD and compares state-of-the-art treatment with routine community care. In a parallel-groups design, 576 children (age, 7-9 years) with ADHD (96 at each site) are thoroughly assessed and randomized to 4 conditions: (1) medication alone, (2) psychosocial treatment alone, (3) the combination of both, (4) or community comparison. The first 3 groups are treated for 14 months and all are reassessed periodically for 24 months. Designers met the following challenges: framing clinically relevant primary questions; defining the target population; choice, intensity, and integration and combination of treatments for fair comparisons; combining scientific controls and standardization with clinical flexibility; and implementing a controlled clinical trial in a nonclinical setting (school) controlled by others. Innovative solutions included extensive decision algorithms and manualized adaptations of treatments to specific needs.

Attention Deficit Disorder with Hyperactivity↗
PubMed1996

Phosphorylation of the herpes simplex virus type 1 tegument protein VP22.

The herpes simplex virus type 1 tegument protein VP22 is known to be highly phosphorylated during infection. Here we show that two electrophoretic forms of VP22 can be identified in infected cell extracts and that this heterogeneity is accounted for by phosphorylation. Furthermore, the nonphosphorylated form of VP22 appears to be specifically incorporated into virions. We also show that the phosphorylated form of VP22 is the only form detected during transient transfection and as such that VP22 can act as a substrate for a cellular kinase. Phospho-amino acid and phospho-peptide analyses of in vivo labeled VP22 were utilized to demonstrate that the phosphorylation profiles of VP22 synthesized during transfection and infection are the same. In both cases VP22 was modified solely on serine residues located in the N-terminal 120 residues of the protein. Moreover, in vitro phosphorylation was utilized to show that the constitutive cellular kinase, casein kinase II, which has four serine consensus recognition sites at the N-terminus of VP22, phosphorylates VP22 in the same manner as observed in vivo. This kinase also phosphorylates VP22 at the N-terminus in intact capsid-tegument structures. Casein kinase II is therefore likely to be the major kinase of VP22 during infection.

Animals↗
PubMed1996

Activation of the erythropoietin (EPO) receptor by bivalent anti-EPO receptor antibodies.

Oligomerization of cytokine receptors including the erythropoietin (EPO) receptor has been advanced as a model for activation. If homodimerization of the EPO receptor activates it, then bivalent antibodies raised to the extracellular domain of the EPO receptor should also homodimerize and activate. Mouse monoclonal antibodies (IgG) raised to the soluble, extracellular domain of the human EPO receptor (EPOR) were found that would stimulate thymidine uptake of an human EPO-dependent cell line, UT-7/EPO. Dose response curves showed bell shapes where activity was low at low and high concentrations. Monovalent (Fab) fragments bound to the receptor but did not stimulate thymidine uptake, which indicates that two antibody binding sites are required for activation. The anti-EPOR antibodies stimulated the formation of burst forming unit erythroid colonies from human CD34(+) cells purified from peripheral blood. This indicates that homodimerization of the EPO receptor by anti-EPOR antibodies is sufficient for both proliferation and differentiation of erythroid progenitor cells and that the constraints on dimerization necessary for activation are rather loose.

Animals↗
PubMed1996

Evidence that biliverdin-IX beta reductase and flavin reductase are identical.

A search of the database shows that human biliverdin-IX beta reductase and flavin reductase are identical. We have isolated flavin reductase from bovine erythrocytes and show that the activity co-elutes with biliverdin-IX beta reductase. Preparations of the enzyme that are electrophoretically homogeneous exhibit both flavin reductase and biliverdin-IX beta reductase activities; however, they are not capable of catalysing the reduction of biliverdin-IX alpha. Although there is little obvious sequence identity between biliverdin-IX alpha reductase (BVR-A) and biliverdin-IX beta reductase (BVR-B), they do show weak immunological cross-reactivity. Both enzymes bind to 2',5'-ADP-Sepharose.

Amino Acid Sequence↗
PubMed1996

Medication treatment strategies in the MTA Study: relevance to clinicians and researchers.

OBJECTIVE: Clinicians have difficulty applying drug research findings to clinical practice, because research protocols use methods different from those used in daily office practice settings. METHOD: To design a medication protocol for a multisite clinical trial involving 576 children with attention-deficit hyperactivity disorder (ADHD) while maintaining relevance to clinical practice, investigators from the NIMH Collaborative Multisite Multimodal Treatment Study of Children with Attention-Deficit/Hyperactivity Disorder (MTA study) developed novel medication strategies. These were designed to work either in a monomodal or multimodal format and to ensure standard approaches are used across diverse sites. Each child randomized to medication (projected N = 288) is individually titrated to his or her "best" methylphenidate dose and has individual ADHD symptoms monitored. Decision rules were developed to guide "best dose" selection, dose changes, medication changes, the management of side effects, and integration with psychosocial treatments. CONCLUSIONS: The MTA study uses a controlled method to standardize the identification of each child's "best" methylphenidate dose in a national, multisite cooperative treatment program. Although the titration protocol is complex, the study's individual dosing approach and algorithms for openly managing ADHD children's medication over time will be of interest to clinicians in office practice.

Adolescent↗
PubMed1995

Megakaryocyte growth and development factor and interleukin-3 induce patterns of protein-tyrosine phosphorylation that correlate with dominant differentiation over proliferation of mpl-transfected 32D cells.

Recently, the ligand for c-mpl, a member of the family of cytokine receptors, was cloned and found to be a physiologic regulator of platelet homeostasis. We report that megakaryocyte growth and development factor (MGDF, thrombopoietin [TPO], c-mpl ligand ) induces differentiation in a majority of mpl-transfected 32D cells, while interleukin (IL)-3 is exclusively mitogenic in this system. MGDF differentiation, as measured by decreased proliferation, changes in cellular morphology, increased adherence, and downregulation of very late antigen (VLA)-4, is dominant over IL-3 proliferation. MGDF induces tyrosine-phosphorylation of mpl, JAK2, SHC, SHPTP-1 (HCP, motheaten) and SHPTP-2 (Syp, PTP-1D) within 30 seconds of stimulation, as well as of vav and MAPK with slightly delayed kinetics. A fraction of mpl and JAK2 is preassociated, and the stoichiometry of this complex is unaltered by cytokine stimulation. After MGDF stimulation, we detect interactions among SHC, grb2, SHPTP-1, SHPTP-2, and the mpl/JAK2 complex. IL-3 induces phosphorylation of the above proteins with the exception of mpl and also causes weak JAK1 phosphorylation. Although similar in composition, the MGDF- and IL-3-induced complexes of signal transducers appear to be assembled in different configurations, especially with respect to SHPTP-2. Both MGDF and IL-3 induce tyrosine phosphorylation of STAT3 (APRF) and STAT5 (MGF), with MGDF favoring STAT3 while IL-3 predominantly causes STAT5 phosphorylation. In addition, some proteins become tyrosine-phosphorylated in response to MGDF only, suggesting that we may have detected differentiation-specific signal transducers. These include a number of high-molecular-weight proteins (140 to 200 kD) and one 28-kD protein that becomes tyrosine-phosphorylated only briefly.

Amino Acid Sequence↗
PubMed1995

Equine herpesvirus 1 gene 12, the functional homologue of herpes simplex virus VP16, transactivates via octamer sequences in the equine herpesvirus IE gene promoter.

The HSV-1 transactivator of immediate-early (IE) gene expression, VP16, has several functional homologues among the alphaherpesviruses which have not yet been extensively studied in relation to their modes of action. To date, nothing is known of the exact sites or mechanism of interaction of the equine herpesvirus type 1 (EHV-1) homologue, the gene 12 protein, with the EHV-1 IE promoter. We show that the gene 12 protein utilises the promoter proximal region of the IE gene to induce activation and identify four potential octamer DNA binding sites within that region. Although there was divergence from its consensus, Oct-1 bound to each of these sites in an in vitro complex formation assay, and in the presence of the gene 12 product a second complex of slower migration, which was also dependent on Oct-1, was detected. When each site was inserted into a basal promoter, two conferred activation by gene 12 with a resulting increase in expression of up to 50-fold compared to basal levels. These results show that, despite the differences between the two proteins, the mechanism of interaction of the gene 12 protein with its target is analogous to that of VP16.

Amino Acid Sequence↗
PubMed1995

Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

Amino Acid Sequence↗
PubMed1995

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling.

The molecular interaction of the Fab fragment of the human monoclonal antibody 3D6, directed against the transmembrane protein gp41 of human immunodeficiency virus (HIV) 1, with its peptide epitope is characterized by a panel of overlapping peptides, a peptide epitope library and molecular modeling techniques. The sequence CSGKLICTTAVPW, corresponding to amino acids 605-617 of gp41, was identified as the best binding peptide (KD = 1 x 10(-8) mol/l). This peptide served as a starting point to prepare a cellulose-bound peptide epitope library in which each residue of the epitope is substituted by all L- and D-amino acids, resulting in 494 epitope peptide variants which were subsequently analyzed for binding 3D6. The library was synthesized to identify residues critical for binding and to obtain information about the molecular environment of the epitope peptide bound to 3D6. Both cysteine residues, as well as isoleucine 6, threonine 8 and proline 12, of the epitope were highly sensitive to substitution. Using the data obtained from the epitope characterization, as well as a low-resolution electron density map of a 3D6 Fab-peptide complex, a 3-D model of the Fab-peptide complex was generated by molecular modeling. The modeling experiments predict binding of the peptide, which is cyclized via the two cysteine residues, to a pocket formed dominantly by the hypervariable loops complementarity determining regions CDR3L, CDR2H and CDR3H.

Amino Acid Sequence↗
PubMed1995

VP16 interacts via its activation domain with VP22, a tegument protein of herpes simplex virus, and is relocated to a novel macromolecular assembly in coexpressing cells.

In addition to its function as a powerful transactivator of viral immediate-early transcription, VP16 is an essential component of the herpes simplex virus (HSV) virion. As such, VP16 is introduced into cells, to effect its function in transactivation, as part of the virus tegument. Here we examine the potential for VP16 protein-protein interactions specific to virus-infected cells and show that VP16 copurifies in a highly enriched fraction with a single major polypeptide which we identify as the virus-encoded structural protein VP22. We further show that in vitro-translated VP22 binds specifically to purified VP16. The activation domain of VP16 was required and largely sufficient for this binding. Mutations within this domain, which disrupt its transactivation function, also affected VP22 binding. Furthermore, we show that while VP16 and VP22 showed distinct patterns of compartmentalization in vivo, coexpression of both proteins resulted in a profound reorganization from their normal locations to a novel macromolecular assembly. The colocalization was also dependent on the activation domain of VP16 but required additional determinants within the N terminus. These results are discussed in the context of VP16 regulation of transcription both early in infection during delivery of tegument proteins and at late times during virus assembly.

Animals↗
PubMed1995

Real-time analysis of the transcriptional regulation of HIV and hCMV promoters in single mammalian cells.

The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.

Animals↗
PubMed1995

Coancestry in apparently sporadic primary pulmonary hypertension.

OBJECTIVE: To examine sporadic cases of primary pulmonary hypertension of coancestry. DESIGN: An epidemiologic study of families of patients with primary pulmonary hypertension. SETTING: A university-affiliated referral population. PARTICIPANTS: Family members of 13 patients with primary pulmonary hypertension. MEASUREMENTS: Family pedigrees involving grandparents, parents, siblings, and children were supplemented by genealogic records. Coefficients of kinship (Ck) were calculated for the patients with primary pulmonary hypertension who demonstrated coancestry and compared with 500 sets of controls drawn at random from genealogic records. RESULTS: Two patients with sporadic primary pulmonary hypertension demonstrated coancestry. The great-great grandfather and great-great grandmother of one patient were the great-grandfather and great-grandmother of the other patient. No other cases of primary pulmonary hypertension were identified in these two families. The CK of the affected individuals (CK = 10.02 x 10(-5)) suggests strongly that the observed relationship did not occur by chance alone. Among 500 random sets of matched controls, only two sets yielded CK of 10.02 x 10(-5) or greater (p = 0.004). Coancestry could not be identified for the other five families of patients with sporadic primary pulmonary hypertension for whom genealogic records were available. CONCLUSIONS: The finding of coancestry in patients with sporadic primary pulmonary hypertension suggests that a genetic basis exists for some patients with apparently sporadic primary pulmonary hypertension. Familial primary pulmonary hypertension may be more common than previously recognized.

Adult↗
PubMed1994

Ultraviolet radiation protection factors for clothing.

There has been considerable interest and activity in Australia during recent years in providing ultraviolet radiation protection for the public and the work force. Many requests from the public and manufacturers have been received by the Australian Radiation Laboratory for the quantification of the protection provided by different articles of clothing. The laboratory has been making spectral measurements of the ultraviolet radiation transmission of materials and fabrics for a number of years and, to this date, have tested and rated > 2,000 samples. The laboratory test methods for making spectral transmission measurements of both dry and wet fabric samples are described along with the radiometric test methods to determine ultraviolet radiation transmission. A scheme to designate the amount of ultraviolet radiation protection of materials using ultraviolet protection factors is outlined and used to compare the results obtained by different test methods. Some typical measurement results are also summarized and presented.

Clothing↗
PubMed1993

Effect of the social environment on alcohol involvement and subjective well-being prior to alcoholism treatment.

This article describes a conceptual model developed to explain abusive use of alcohol and reports results of an initial test of that conceptualization. General social support is hypothesized to affect the level of subjective well-being, while alcohol-relevant social support affects the degree of alcohol involvement. A cross-sectional test of two models derived from this formulation was made using data from 148 alcoholic clients entering treatment at a private non-profit psychiatric facility. Results indicate that a model in which no direct relationship between alcohol involvement and subjective well-being is specified provides a more parsimonious explanation of interrelationships at the time of treatment entry. Alcohol involvement is explained by alcohol-relevant affiliative and instrumental support (albeit weakly), and subjective well-being is explained by general affiliative and instrumental social support. There is virtually no interrelationship between alcohol involvement and subjective well-being once the support variables are taken into account. The findings indicate that treatment should more often incorporate others from a client's social network, using significant others first to provide general social support and later alcohol-relevant support as well. Further analyses involving samples with more heterogeneity in levels of social investment and exploring the utility of these variables for treatment matching categorization decisions are warranted.

Adaptation, Psychological↗
PubMed1990

Monitoring mutagenicity in urine and peripheral blood lymphocytes of pharmacists occupationally exposed to anticancer drugs.

Two approaches to mutagenicity monitoring were compared for estimation of possible absorption of cytotoxic drugs by a group of six pharmacists at Auckland Hospital. Blood samples were taken before and after a three week duty roster, from age and sex matched negative controls, and from a matched group of patients on chemotherapy. Peripheral blood lymphocytes were fixed, stained and analysed for sister chromatid exchange (SCE). Urine samples were collected from the pharmacists on the morning following their last day on cytotoxic dispensing duty, as well as from positive and negative controls. Samples were concentrated and analysed for mutagenicity in the bacterial Ames test. Comparison of negative control groups with the pharmacists using either of these test measures gave no evidence for significant absorption of cytotoxics by the pharmacist group as a whole, although values for the positive control groups were raised significantly in a similar comparison. The value for SCE of one pharmacist however, was elevated in the second sample (after three weeks dispensing duty) when compared with the first sample (before duty) and the urine analysis also suggested that this individual might have absorbed some mutagens. The results permit a comparison of these techniques as a means of ongoing monitoring of similar populations within New Zealand.

Antineoplastic Agents↗
PubMed1989

Protein engineering and design.

Rapid advances in site-directed mutagenesis and total gene synthesis combined with new expression systems in prokaryotic and eukaryotic cells have provided the molecular biologist with tools for modification of existing proteins to improve catalytic activity, stability and selectivity, for construction of chimeric molecules and for synthesis of completely novel molecules that may be endowed with some useful activity. Such protein engineering can be seen as a cycle in which the structures of engineered molecules are studied by X-ray analysis and two-dimensional nuclear magnetic resonance. The results are used in the improvement of the design by using knowledge-based procedures that exploit facts, rules and observations about proteins of known three-dimensional structure.

Amino Acid Sequence↗