Showing papers by "Kettering University published in 1997"

Rapid Plasma Membrane Anchoring of Newly Synthesized p59 fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

Abstract: The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59 fyn . Metabolic labeling of transfected COS or NIH 3T3 cells with [ 35 S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH 2 -terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gα o -, Gα s -, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.

Basic fibroblast growth factor (bFGF) upregulates the expression of bcl-2 in B cell chronic lymphocytic leukemia cell lines resulting in delaying apoptosis.

Abstract: To evaluate the function of HTLV-I env-pX gene in vivo, we developed two lines of transgenic rats (env-pX rats) that expressed env-pX gene products, under control of own LTR promotor. In various tissues of the rats, env and pX mRNAs were constitutively expressed, irrespective of age. At age 5 weeks, swelling of the bilateral ankle joints histologically showing synovial lining hyperplasia, severe chronic inflammation, erosion of the joint cartilage, and bone destruction with pannus formation began to develop in these env-pX rats. These histologic features resemble those of rheumatoid arthritis (RA) in man. High titered rheumatoid factors and low anti-dsDNA antibodies and hyper-gamma globulinemia were detected. Necrotizing arteritis resembling polyarteritis nodosa, polymyositis, myocarditis and Sjogren syndrome-like sialoadenitis developed, together with RA-like arthritis even in one individual animal. Thymic atrophy with low body weight was also observed. The evidence indicates that env-pX rats appear to be suitable animal models for elucidating pathogenetic mechanisms involved in not only HTLV-I related diseases but also various collegen vascular and autoimmune diseases of unknown etiology in man.

Epidemiology of psychiatric disorders in elderly compared with younger adults with learning disabilities.

Abstract: BACKGROUND The literature regarding psychiatric illness among elderly people with learning disabilities is limited and conflicting because of methodological differences. There have been no recent studies comparing psychiatric epidemiology between younger and older adults with learning disabilities, using the same methodology and definitions. METHOD Comprehensive psychiatric examination using a semi-structured rating scale was undertaken on everyone with learning disabilities, aged 65 years or over (n = 134), living in a defined geographical area. Comparison was made with a randomly selected control group of adults with learning disabilities aged 20-65 years (n = 73) drawn from the same geographical area. RESULTS Elderly people with learning disabilities have a greater prevalence of psychiatric morbidity than younger controls (68.7 v. 47.9%). Rates for depression and anxiety disorders are high, and dementia is common: there are equal rates for schizophrenia/delusional disorders, autism and behaviour disorders in the two groups. CONCLUSIONS The higher psychiatric morbidity among elderly (compared with younger) people with learning disabilities has not previously received adequate recognition. This warrants further investigation by service planners and clinicians.

Regulation of Hematopoiesis by Microvascular Endothelium

Abstract: The bone marrow microenvironment is a complex three dimensional structure where hematopoietic stem cells proliferate, mature, migrate into the sinusoidal space, and enter the circulation in an exquisitely regulated fashion. Stromal cells within the BM microenvironment provide a suitable environment for self-renewal, proliferation and differentiation of hematopoietic stem cells. Within the hematopoietic microenvironment, whether it is embryonic yolk sac, fetal liver, or adult bone marrow, microvascular endothelium not only acts as a gatekeeper controlling the trafficking and homing of hematopoietic progenitors, but also provides cellular contact and secretes cytokines that allows for the preservation of the steady state hematopoiesis. Recently, homogenous monolayers of bone marrow endothelial cells (BMEC) have been isolated and cultivated in tissue culture. Long-term coculture studies have shown that BMEC monolayers are unique type of endothelium and can support long-term proliferation of hematopoietic progenitor cells particularly megakaryocytic and myeloid progenitor cells by constitutive elaboration of lineage-specific cytokines such as G-CSF, GM-CSF, M-CSF, Kit-ligand, IL6, FLK-2 ligand, and leukemia inhibitory factor. Direct cellular contact between hematopoietic progenitor cells and BMEC monolayers through specific adhesion molecules including beta1, beta2 integrins and selectins play a critical role in trafficking and possibly proliferation of hematopoietic stem cells. Dysfunction of microvascular endothelial cells within the hematopoietic microenvironment may result in stem cell disorders and progression to aplastic anemias, and contribute to graft failure during bone marrow transplantation. Further studies on the role of microvascular endothelium in the regulation of hematopoietic stem cell homing and proliferation may enhance our understanding of the pathophysiology of stem cell and leukemic disorders.

Phylogeny of mRNA capping enzymes

Abstract: The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structure–function analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme. The results also allowed us to identify the capping enzyme of Caenorhabditis elegans. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.

Implantable sensor employing an auxiliary electrode

Abstract: An implantable sensor (32) comprising a biocompatible electroconductive case (42) which houses a measuring electrode (34), a reference electrode (36), an auxiliary electrode (38), and an electronic circuit (40) for measuring the response of the measuring electrode where the measuring electrode, reference electrode and auxiliary electrode are not in direct contact with one another is provided. The implantable sensor of the present invention is particularly useful as a glucose sensor, especially when an enzyme-containing membrane is included in the measuring electrode.

High prevalence of dementia among people with learning disabilities not attributable to Down's syndrome.

Abstract: Background. For many years, it has been known that dementia can occur in people with learning disabilities, but there have been few research studies. Studies that do quote rates for dementia show these to be high, but this important fact has received remarkably little attention.Method. Comprehensive psychiatric and medical assessments were undertaken on the whole population (ascertained as far as is possible) of people with learning disabilities aged 65 years and over living in Leicestershire, UK (N=134), in order to ascertain rates of DCR defined dementia, and associated factors.Results. Dementia was diagnosed in 21·6%, against an expected prevalence of 5·7%, for a group with this age structure. The rate of dementia increased in successive age cohorts: 15·6% aged 65–74 years; 23·5% aged 65–84 years; and 70·0% aged 85–94 years. People with dementia tended to be older, female, with more poorly controlled epilepsy, a larger number of additional physical disorders, less likely to be smokers and had lower adaptive behaviour scores than did the elderly people without dementia. They were more likely to live in health service accommodation.Conclusions. Dementia occurs at a much higher rate among elderly people with learning disabilities than it does among the general population; this is independent of the association between dementia and Down's syndrome. Whether this relates aetiologically to genetics, lack of brain ‘reserve’ or history of brain damage is yet to be determined.

Differential toxicity of camptothecin, topotecan and 9-aminocamptothecin to human, canine, and murine myeloid progenitors (CFU-GM) in vitro

Abstract: Purpose: 20(S)-Camptothecin (CAM), topotecan (TPT, active ingredient in Hycamtin) and 9-amino-20(S)-camptothecin (9AC) are topoisomerase I inhibitors that cause similar dose-limiting toxicities to rapidly renewing tissues, such as hematopoietic tissues, in humans, mice, and dogs. However, dose-limiting toxicity occurs at tenfold lower doses in humans than in mice. The purpose of the current study was to determine whether hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to these compounds that is evident in vivo. Methods: Drug-induced inhibition of in vitro colony formation by a myeloid progenitor in human, murine, and canine marrow colony-forming unit-granulocyte/macrophage (CFU-GM) provided the basis for interspecies comparisons at concentrations which inhibited colony formation by 50% (IC50) and 90% (IC90). Results: Murine IC90 values were 2.6-, 2.3-, 10-, 21-, 5.9-, and 11-fold higher than human values for CAM lactone (NSC-94600) and sodium salt (NSC-100880), TPT (NSC-609699), and racemic (NSC-629971), semisynthetic and synthetic preparations (NSC-603071) of 9AC, respectively. In contrast, canine IC90 values were the same as, or lower than, the human IC90 values for all six compounds. Conclusions: The greater susceptibility of humans and dogs to the myelotoxicity of camptothecins, compared to mice, was evident in vitro at the cellular level. Differential sensitivity between murine and human myeloid progenitors explains why the curative doses of TPT and 9AC in mice with human tumor xenografts are not achievable in patients. Realizing the curative potential of these compounds in humans will require the development of therapies to increase drug tolerance of human CFU-GM at least to a level equal to that of murine CFU-GM. Because these interspecies differences are complicated by species-specific effects of plasma proteins on drug stability, not all in vitro assay conditions will yield results which can contribute to the development of such therapies.

c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.

Abstract: In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.

Single chain FV polynucleotide or peptide constructs of anti-ganglioside GD2 antibodies, cells expressing same and related methods

Abstract: Recombinant antibody constructs comprise the variable regions of the heavy and light chains of anti-G D2 antibodies. These antibody constructs may be coupled to a label such as a radiolabel or to a protein such as streptavidin or pro-drug converting enzymes for use in imaging or therapeutic applications. The antibody constructs may also be transduced into T cells to produce populations of T cells which target G D2 -producing tumor cells.

Convergent total synthesis of a tumour-associated mucin motif.

Abstract: Synthetic glycoconjugates that mimic cell-surface tumour antigens (glycolipids or glycoproteins with unusual carbohydrate structural motifs) have been shown to trigger humoral responses in murine and human immune systems1,2,3 This raises the exciting possibility of inducing active immunity with fully synthetic carbohydrate vaccines, particularly if vaccine compounds can be synthesized that resemble the surface environment of transformed cells even more closely Glycopeptides seem particularly suitable for this purpose In contrast to most glycolipids and thecarbohydrates themselves, glycopeptides bind to major histocompatibility complex molecules, and, in favourable cases, can stimulate T cells and lead to the expression of receptors that recognize the carbohydrate part of a glycopeptide with high specificity4,5,6,7,8 The preparation of glycopeptides and glycoproteins remains, however, a difficult challenge9,10,11,12: earlier synthesis methods have been inefficient, and established cloning approaches that allow engineering of global glycopatterns produce only heterogeneous glycoproteins13 Here we report an efficient strategy of the synthesis of tumour-associated mucin glycopeptides with clustered trisaccharide glycodomains corresponding to the (2,6)-sialyl T antigen Our approach involves construction of the complete glycodomain in the first stage, followed by convergent coupling to amino acid residues and subsequent incorporation of the glycosyl amino acid units into a peptide chain This general strategy allows the assembly of molecules in which selected glycoforms can be incorporated at any desired position of the peptide chain The resultant fully synthetic O-linked glycopeptide clusters are the closest homogeneous mimics of cell-surface mucins at present available, and so are promising compounds for the development of anticancer vaccines

Clinical study of the effects of age on the physical health of adults with mental retardation

Abstract: Physical disorders and pharmacotherapy for all 134 people with mental retardation ages 65 years and over living in Leicestershire, United Kingdom, were examined. Results were compared with a randomly selected group of 73 younger adults with mental retardation. Group comparisons revealed higher rates of urinary incontinence, immobility, hearing impairments, arthritis, hypertension, and cerebrovascular disease among the older group. The younger group had higher rates of dermatological disorders; congenital heart disease; ear, nose, and throat (ENT) disorders; and neurological disorders (excluding Parkinson disease). The older group took more drugs for physical illness. The effect of ageing on physical morbidity outweighs the effect of people with more severe mental retardation dying younger: Older people with mental retardation have significant physical health needs.

Synergistic mechanisms by which sirolimus and cyclosporin inhibit rat heart and kidney allograft rejection

Abstract: The studies presented herein examined the mechanism(s) whereby sirolimus (SRL) and cyclosporin (CsA) act synergistically to block allograft rejection. Combination index (CI=1 reflects additive, CI<1 antagonistic, and CI<1 synergistic, effects) analysis documented potent synergism between SRL and CsA to block allograft rejection. Combinations of the two drugs produced synergistic prolongation of heart (CI=0.001–0.2) or kidney (CI=0.03–0.5) allograft survival at SRL/CsA ratios ranging from 1:12.5 to 1:200. Pharmacokinetic analysis of the individual drugs showed that CsA does not affect the blood levels of SRL, and SRL mildly increases the levels of CsA in SRL/CsA-treated rats. Quantitative polymerase chain reaction analysis was used to document that both subtherapeutic (1.0 mg/kg) and therapeutic (2.0 or 4.0 mg/kg) CsA doses inhibited the expression of interferon-gamma (IFN-γ) (P<0.03) and IL-2 (P<0.003) mRNA produced by T helper (Th) 1 cells, as well as IL-10 (P<0.001), but not IL-4 (NS) mRNA produced by Th2 cells. Contrariwise, all tested SRL doses (0.02, 0.04 or 0.08 mg/kg) did not affect cytokine mRNA expression. However, heart allografts from rat recipients treated with synergistic SRL/CsA doses displayed reduced levels of IFN-γ (P<0.01), IL-2 (P<0.001) and IL-10 (P<0.001) mRNA. Thus, because subtherapeutic doses of CsA reduce Th1/Th2 activity, thereby facilitating the inhibition of signal transduction by low does of SRL, the two agents act synergistically to inhibit allograft rejection.

Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1.

Abstract: We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.

Analysis of CD28 cytoplasmic tail tyrosine residues as regulators and substrates for the protein tyrosine kinases, EMT and LCK.

Abstract: The CD28 cell surface receptor provides an important costimulatory signal for T cells necessary for their response to Ag. Early events in CD28 signaling include recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase) and activation of the protein tyrosine kinases (PTKs), LCK and EMT. Recruitment and activation of PI3-kinase is known to be dependent upon phosphorylation of tyrosine 173 of the CD28 cytoplasmic tail contained within a YMNM motif. By contrast, little is known of which residues of the CD28 tail, including tyrosines, are required for the activation of PTKs. To address this we studied the ability of truncation mutants and tyrosine to phenylalanine substitution mutants of the CD28 cytoplasmic tail to activate LCK and EMT in Jurkat T leukemia cells. Our results indicate that 1) activation of EMT is partially dependent upon tyrosine 173 of the CD28 tail, although it does not require PI3-kinase activation; 2) activation of LCK is independent of CD28 cytoplasmic tail tyrosine residues; and 3) elements sufficient for the activation of both kinases are contained within the first half of the tail. In addition we studied the CD28 tail as a substrate for both PTKs in in vitro kinase assays. We demonstrate that EMT can phosphorylate all four tyrosines of the CD28 tail, in contrast to LCK, which phosphorylates only tyrosine 173. Together with evidence that in vivo, tyrosines other than tyrosine 173 become phosphorylated following CD28 stimulation, this finding suggests that, like LCK, one function of EMT during CD28 signaling is phosphorylation of the receptor.

Method and reagents for genetic immunization

Abstract: DNA vaccines which incorporate genetic sequences encoding sorting signals which direct an expressed antigen to a specific cellular organelle facilitate loading of the antigen onto a Class I or Class II MHC molecule for immune presentation. These vaccines are a nucleic acid construct of a genetic sequence encoding a protein or peptide antigen and a sorting signal which will direct expressed antigen to the ER or endosomal-lysosomal compartments within the cell. The resulting constructs can be used as naked DNA vaccines, packaged in liposomes, or coated onto colloidal gold particles. The construct might also be delivered in an expression vector which is expressed in cells of the organism being immunized.

A highly convergent total synthetic route to glycopeptides carrying a high-mannose core pentasaccharide domain N-linked to a natural peptide motif

Abstract: N-Linked glycopeptides were synthesized by condensation of a highmannose anomeric amine bearing a pentasaccharide with aspartic-acid-containing tri- and pentapeptides through the agency of IIDQ. The pentasaccharide portion, corresponding to the „core” region of all asparagine-linked glycoproteins, was assembled by means of glycal-derived thioethyl donors and glycal acceptors. The central mannose residue was established by inversion of the C2 hydroxyl of a glucosyl precursor in the pentasaccharide. The protecting-group scheme employed allows the extension of the pentasaccharide through the terminal mannose units. While a fully convergent coupling of the high-mannose carbohydrate to the peptide domain has thus been accomplished for the first time with a fully synthetic sugar, the stereochemical integrity of the anomeric center of the carbohydrate domain was not maintained and a mixture of glycopeptides was obtained.

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.

Abstract: Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.

Total synthesis of the potential anticancer vaccine KH-1 adenocarcinoma antigen

Abstract: Human tumours are often marked by the expression of unusual carbohydrate structural motifs. These carbohydrate domains are manifested as cell-surface bound glycolipids or glycoproteins. This raises the possibility of using cell-free equivalents of these domain compounds, obtained by total synthesis with a view towards triggering some level of immune response. In fact, the serum of mice immunized with fully synthetic compounds that mimic cell-surface tumour antigens has already been shown to recognize pertinent human cancer cell lines. Further advances in this field depend critically on the availability of these tumour-associated carbohydrate antigens which cannot be readily isolated from natural sources in sufficient quantities. Here we present the successful total synthesis of an adenocarcinoma antigen, KH-1, and of a bioconjugatable analogue which can bind to a carrier protein. These results illustrate the capabilities of oligosaccharide synthesis for reconstructing the challenging structural motifs characteristic of carbohydrate antigens, and thereby open up new possibilities for the development of anticancer vaccines.

Combinations of pkc inhibitors and therapeutic agents for treating cancers

Abstract: This invention provides methods for screening a modulating agent which when combined with antitumor therapeutic agent increases apoptosis in tumor cells. This invention also provides methods for screening antitumor therapeutic agents suitable for combination therapy with protein kinase C inhibitors capable of potentiating apoptosis in tumor cells. This invention further provides different combination therapies comprising the specific protein kinase C inhibitors and the antitumor therapeutic agents.