PubMed Health⌕ Search

Biomedical subjects

E McGlynn

Publications and source records attributed to E McGlynn.

17 recordsLinked to original sources

Services to families of adults with schizophrenia: from treatment recommendations to dissemination.

OBJECTIVE: Data from the Schizophrenia Patient Outcomes Research Team project were examined to determine the extent to which families of adults with schizophrenia receive services and whether training staff in the provision of family services increases service availability. METHODS: For patients with a diagnosis of schizophrenia, paid claims for family therapy were identified in 1991 in a nationally representative sample of Medicare data and one state's Medicaid data. In a field study in two states, 530 patients were asked about services received by their families. A quasiexperimental dissemination of a family intervention was done at nine agencies; staff at four agencies received a standard didactic presentation, and staff at five received that standard presentation paired with intensive training. RESULTS: In the representative national Medicare sample of 15,425 persons with schizophrenia, .7 percent (N=108) had an outpatient claim for family therapy. This figure was 7.1 percent in the Medicaid sample of 5,393 persons with schizophrenia in one state. Of the 530 patients in the field study who reported having contact with their families, 159 (30 percent) reported that their families had received information, advice, or support about their illness, and 40 (8 percent) responded that their families had attended an educational or support program. At the four agencies where staff received only didactic training, no changes in family services were found after one year. Three of the five agencies where staff participated in intensive training enhanced their family services. CONCLUSIONS: A minority of families of persons with schizophrenia receive information about the illness from providers. Implementation of model family interventions is possible with considerable technical assistance. A gap exists between best practices and standard practices for families of persons with schizophrenia.

Adult↗

Retractions.

Explore the source record for details and available documents.

Retraction Notice↗

Improving the quality of healthcare for children: implementing the results of the AHSR research agenda conference.

OBJECTIVE: To describe the rationale, development, content, and results of the AHSR-sponsored conference on developing a research agenda focused on improving the quality of care for children. DATA SOURCES AND METHODS: Planning documents, background papers, and conference proceedings. PRINCIPAL FINDINGS: The conference developed the research agenda focused on (1) monitoring the health of children; (2) evaluating the efficacy and effectiveness of health services for children; (3) assessing the quality of healthcare provided to children; (4) improving the quality of healthcare within health systems; (5) assessing the performance of community systems for children; (6) exploring the impact of different financial incentives on the provision of pediatric healthcare; and (7) developing and disseminating clinical practice guidelines and other information to physicians, families, and consumers. Specific issues and research questions in each area are also presented. Strategies for implementing the research agenda are presented and include: (1) expanding the child health services research workforce; (2) developing child healthcare quality improvement research centers; (3) conducting research in specific high-priority areas; (4) focusing research on improving the health of vulnerable populations; (5) improving child health data and collection systems at the national level; (6) developing better community health monitoring for children; (7) building and supporting research networks and a consortium of research users; and (8) developing a coordinated interagency federal effort to advance this agenda and to provide accountability for its completion. CONCLUSION: The proposed research agenda should be a national priority so that all Americans can be assured that children are receiving the best quality of care that the United States can provide.

Child↗

Localization of the heparin-binding site of glia-derived nexin/protease nexin-1 by site-directed mutagenesis.

Recombinant rat glia-derived nexin was expressed in insect cells using the baculovirus system. The kinetics for the inhibition of thrombin by this recombinant material were indistinguishable from those observed with natural glia-derived nexin and recombinant nexin expressed in yeast. In addition, the dependence of the rate of inactivation on the concentration of heparin was similar for the three preparations. At the optimal heparin concentration, the association rate constant was 330-fold higher than that observed in the absence of heparin. A putative heparin-binding site is found in glia-derived nexin between residues 71 and 86; heparin-binding sites are found in homologous regions of antithrombin III and heparin cofactor II. Lysines in this region were mutated to glutamates, and the kinetics for the inhibition of thrombin by mutant proteins were determined. Concurrent mutation of all seven lysines in this region (residues 71, 74, 75, 78, 83, 84, and 86) did not affect the rate constant for the association of glia-derived nexin with thrombin in the absence of heparin, but it resulted in complete loss of the heparin acceleration of the rate of association. Mutations of residues 83, 84, and 86 together also caused a marked decrease in the acceleration by heparin of the reaction between glia-derived nexin and thrombin. These results support the hypothesis that the heparin-binding sites of glia-derived nexin, antithrombin III, and heparin cofactor II are found in homologous regions of the molecules. Heparin was also found to potentiate the ability of wild-type glia-derived nexin to inhibit the thrombin-induced retraction of neurites from neuroblastoma NB2a cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

4,5-Dianilinophthalimide: a protein-tyrosine kinase inhibitor with selectivity for the epidermal growth factor receptor signal transduction pathway and potent in vivo antitumor activity.

Deregulated signal transduction via the epidermal growth factor receptor (EGF-R) family of protein-tyrosine kinase growth factor receptors is associated with proliferative diseases. We describe a class of compounds (4,5-dianilinophthalimides) that inhibit the EGF-R protein-tyrosine kinase in vitro with high selectivity. In cells, 4,5-dianilinophthalmide selectively inhibited both ligand-induced EGF-R and p185c-erbB2 autophosphorylation and c-fos mRNA induction. Antitumor activity could be demonstrated in vivo against xenografts of the A431 and SK-OV-3 tumors, which overexpress the EGF-R and p185c-erbB2, respectively. In contrast, a platelet-derived growth factor-driven tumor was not inhibited by 4,5-dianilinophthalimide, which is compatible with its cellular selectivity and hypothesized mechanism of action. No overt cumulative toxicity was observed during treatment even though high efficacy was observed, indicating a good therapeutic window. 4,5-Dianilinophthalimides may offer therapeutic agents for the treatment of hyperproliferative diseases that overexpress EGF-R family protein-tyrosine kinases or their ligands.

3T3 Cells↗

Existent T-cell and antibody immunity to HER-2/neu protein in patients with breast cancer.

The HER-2/neu protooncogene is amplified and overexpressed in 20-40% of invasive breast cancers. HER-2/neu protein overexpression is associated with aggressive disease and is an independent predictor of poor prognosis in several subsets of patients. The protein may also be related to cancer formation, with overexpression being detectable in 50-60% of ductal carcinomas in situ. It has been suggested that it might be possible to develop specific T-cell therapy directed against proteins involved in malignant transformation. One question is whether normal proteins that are overexpressed are appropriate targets for therapeutic immune attack. This report demonstrates that some patients with HER-2/neu-positive breast cancers have both existent CD4+ helper/inducer T-cell immunity and antibody-mediated immunity to HER-2/neu protein. Initial studies performed on 20 premenopausal breast cancer patients identified antibodies to HER-2/neu in 11 individuals. Similar antibody responses have been found in some normal individuals. The patient with the greatest antibody response was studied in detail. In addition to a humoral immune response this patient had evidence of a significant proliferative T-cell response to the HER-2/neu protein and peptides. Similar T-cell responses have been detected in additional patients. It has been assumed that patients would be immunologically tolerant to HER-2/neu as a self-protein and that immunity might be difficult to generate. If immunity could be generated, the result might be destructive autoimmunity. The current data support the notion that HER-2/neu-specific immunity might be used in therapy without destroying normal tissue but also raises questions as to the role of existent immunity in immune surveillance and cancer progression.

3T3 Cells↗

Active baculovirus recombinant p70s6k and p85s6k produced as a function of the infectious response.

Rat p70s6k and p85s6k have been expressed in baculovirus recombinants propagated in Sf9 insect cells. Surprisingly, both recombinant isoforms were active without coinfection of other kinases which lie upstream in the signaling pathway. Treatment of either recombinant form with phosphatase 2A leads to immediate inactivation in the absence of phosphatase inhibitors. Further studies show that the same four major Ser/Thr-Pro sites associated with p70s6k activation following mitogenic stimulation in vivo are also the four major sites phosphorylated in both the p70s6k and p85s6k during the infection process. It is proposed that the production of phosphorylated and activated recombinant p70s6k and p85s6k is due to activation of a host cell signaling pathway which is triggered by viral infection. In support of this hypothesis, wild-type virus-, but not mock-infected cells, exhibit the multiple phosphorylation of a ribosomal protein which migrates similar to ribosomal protein S6 on two-dimensional-polyacrylamide gels and extracts from these same cells contain elevated levels of S6 kinase activity.

Animals↗

Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase.

To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.

Amino Acid Sequence↗

Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase. Modulation of activity by multiple factors.

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.

Angiotensin II↗

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity.

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.

Enzyme-Linked Immunosorbent Assay↗

Expression and partial characterization of rat protein kinase C-delta and protein kinase C-zeta in insect cells using recombinant baculovirus.

Expression of rat protein kinase C-delta (PKC-delta) and PKC-zeta in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype-specific antisera. Although the PKC-zeta cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC-delta were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC-alpha. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC-zeta. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC-delta or PKC-zeta when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC-delta and PKC-zeta. In contrast to PKC-zeta, the PKC-delta enzyme activity phosphorylated MBP or histone in a phosphatidylserine-(PS)/diacylglycerol(DG)-dependent manner, albeit not to the same extent as PKC-alpha. Lack of stimulation of the enzyme activity of PKC-zeta by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC-zeta, whereas several insect cell proteins were phosphorylated by PKC-delta in a PS/DG-dependent manner, including a protein of 78 kD. Our data demonstrate that the 76 kD PKC-zeta, in contrast to PKC-delta, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS or PS/DG. In addition, staurosporine was about 2-4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC-delta and PKC-zeta when compared to PKC-alpha.

Alkaloids↗

Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.

Baculoviridae↗

S-cyclophilin. New member of the cyclophilin family associated with the secretory pathway.

Cyclophilin is an abundant and ubiquitous cytosolic protein that is conserved throughout evolution from man to bacteria. It is the target of the immunosuppressive drug cyclosporin A. Cyclophilin has peptidyl-prolyl cis/trans-isomerase activity, and it accelerates protein folding in vitro, suggesting that it might be involved in the folding of cytosolic proteins. We describe a novel cyclophilin-like protein, S-cyclophilin, in the chick. Analysis of S-cyclophilin cDNA revealed the presence of a signal sequence followed by an open reading frame coding for a protein very similar to cytosolic cyclophilin, except for the presence of unique additional short amino acid segments at the N and C termini of the protein. S-Cyclophilin mRNA was abundant and present in all embryonic chick tissues tested. Cyclophilin and S-cyclophilin are coded by separate genes in the chick genome. Recombinant S-cyclophilin was expressed in insect cells by means of the baculovirus system. Pulse-chase experiments revealed that a significant fraction of newly synthesized recombinant S-cyclophilin was rapidly secreted into the culture medium. Our findings indicate that cyclophilins are associated with most if not all intra- and extracellular compartments and suggest that enzyme-assisted conformational conversions in proteins might also take place in post-endoplasmic reticulum compartments, possibly including the extracellular space.

Amino Acid Isomerases↗

Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.

Various derivatives of thiazolidine-diones have been identified as tyrosine protein kinase inhibitors. The epidermal growth factor (EGF) receptor kinase and c-src kinase were inhibited in vitro with IC50 values in the range of 1-7 microM. The v-abl tyrosine protein kinase was not inhibited by thiazolidine-diones. Inhibition was found to be specific for tyrosine protein kinases. Inhibition of serine/threonine protein kinases was not observed. The active derivatives were shown to inhibit EGF-induced receptor autophosphorylation, either in vitro or in intact cells, and were also found to inhibit growth of the EGF-dependent BALB/MK and A431 cell lines (IC50 1-3 microM). Growth of the interleukin-3-dependent myeloid cell line FDC-P1 was inhibited with equal efficiency. Thus, in these cell lines, members of the c-src kinase family are also potential targets for inhibition by the compounds.

Animals↗

Evaluation of renal transplant rejection by duplex Doppler examination: value of the resistive index.

The increasing use and availability of renal transplantation has resulted in a demand for noninvasive methods to study possible complications. One of the most serious adverse reactions is acute rejection, a possibly reversible cause of transplant failure if treated promptly. Differentiation from other causes of acute renal failure frequently is difficult, and the lack of specificity in many imaging studies has been troublesome. Eighty-one patients with renal transplants, including 41 with acute rejection, were examined. Duplex Doppler examination of the intrarenal arteries and a simplified formula, the resistive index ([peak systolic frequency shift--lowest diastolic frequency shift]/[peak systolic frequency shift]), were used to diagnose rejection. With a resistive index greater than 0.90, a 100% positive predictive value was obtained for the diagnosis of acute rejection. A value less than 0.70 was unlikely to be rejection (negative predictive value, 94%). This approach uses a simple analysis of the waveform. Use of a duplex Doppler examination and the formula described here appears to be an accurate method for the detection of acute rejection and for the differentiation of acute rejection from the various other causes of acute renal failure.

Acute Kidney Injury↗