Showing papers by "Novartis published in 1995"

A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules

Abstract: We present the derivation of a new molecular mechanical force field for simulating the structures, conformational energies, and interaction energies of proteins, nucleic acids, and many related organic molecules in condensed phases. This effective two-body force field is the successor to the Weiner et al. force field and was developed with some of the same philosophies, such as the use of a simple diagonal potential function and electrostatic potential fit atom centered charges. The need for a 10-12 function for representing hydrogen bonds is no longer necessary due to the improved performance of the new charge model and new van der Waals parameters. These new charges are determined using a 6-31G* basis set and restrained electrostatic potential (RESP) fitting and have been shown to reproduce interaction energies, free energies of solvation, and conformational energies of simple small molecules to a good degree of accuracy. Furthermore, the new RESP charges exhibit less variability as a function of the molecular conformation used in the charge determination. The new van der Waals parameters have been derived from liquid simulations and include hydrogen parameters which take into account the effects of any geminal electronegative atoms. The bonded parameters developed by Weiner et al. were modified as necessary to reproduce experimental vibrational frequencies and structures. Most of the simple dihedral parameters have been retained from Weiner et al., but a complex set of 4 and yj parameters which do a good job of reproducing the energies of the low-energy conformations of glycyl and alanyl dipeptides has been developed for the peptide backbone.

Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease

Abstract: Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

The biology of hematopoietic stem cells

Abstract: Hematopoietic stem cells (HSC) are the only cells in the blood-forming tissues that can give rise to all blood cell types and that can self-renew to produce more HSC. In mouse and human, HSC represent up to 0.05% of cells in the bone marrow. HSC are almost entirely responsible for the radioprotective and short- and long-term reconstituting effects observed after bone marrow transplantation. The subsets of HSC that give rise to short-term vs long-term multilineage reconstitution can be separated by phenotype, demonstrating that the fates of HSC are intrinsically determined. Here we review the ontogeny and biology of HSC, their expression of fate-determining genes, and the clinical importance of HSC for transplantation and gene therapy.

Plaque-associated expression of human herpesvirus 6 in multiple sclerosis

Abstract: Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in > 70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.

Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates

Abstract: Compounds of the formulae I and Ia and their racemates ##STR1## in which A is --CH 2 -- or --CH 2 CH 2 --, R 1 is hydrogen or a protective group, R 2 is hydrogen or a protective group or a radical forming a phosphorus-containing nucleotide bridge group and B is a purine or pyrimidine radical or an analogue thereof, can be used as antiviral active ingredients or for the preparation of biologically active oligonucleotides.

Ein neuer Zugang zu 2′-O-Alkylribonucleosiden und Eigenschaften deren Oligonucleotide

Abstract: A New Access to 2′-O-Alkylated Ribonucleosides and Properties of 2′-O-Alkylated Oligoribonucleotides A general access to 2′-O-alkylated ribonucleosides using the key intermediate 5 is presented. The incorporation of 2′-O-‘ethyleneglycol’- and 2′-O-‘glycerol’-substituted (i.e., 2′-O-(2-hydroxyethyl)- and 2′-O-(2,3-dihydroxypropyl)-substituted) ribonucleosides into oligonucleotides affords a new generation of oligonucleotides with high affinity for RNA, high specificity, and increased nuclease resistance.

Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene

Abstract: Systemic acquired resistance (SAR) is an inducible plant response to infection by a necrotizing pathogen. In the induced plant, SAR provides broad-spectrum protection against not only the inducing pathogen, but also against other, unrelated pathogens. Both salicylic acid (SA) and SAR-gene expression have been implicated as playing important roles in the initiation and maintenance of SAR. Here, we describe the characterization of transgenic Arabidopsis plants that express the bacterial nahG gene encoding salicylate hydroxylase, an enzyme that can metabolize SA. Strong, constitutive expression of this gene prevents pathogen-induced accumulation of SA and the activation of SAR by exogenous SA. We show that SAR in Arabidopsis can be induced by inoculation with Pseudomonas syringe pv. tomato against infection by a challenge inoculation with Peronospora parasitica. This response is abolished in transgenic, nahG-expressing Arabidopsis, but not in ethylene-insensitive mutants. These experiments support the critical role of SA in SAR and show that ethylene sensitivity is not required for SAR induction. The NahG Arabidopsis plants will be important for future studies aimed at understanding the role of SA in plant disease resistance mechanisms.

A multicenter, placebo-controlled, randomized, double-blind, prospective trial of prophylactic ursodiol for the prevention of gallstone formation following gastric-bypass-induced rapid weight loss.

Abstract: Background Previous studies have documented a high incidence of gallstone formation following gastric-bypass (GBP)-induced rapid weight loss in morbidly obese patients. This study was designed to determine if a 6-month regimen of prophylactic ursodiol might prevent the development of gallstones. Methods A multicenter, randomized, doubleblind, prospective trial evaluated 3 oral doses of ursodiol: 300, 600, and 1,200 mg versus placebo beginning within 10 days after surgery and continuing for 6 months or until gallstone development, for patients with a body mass index (BMI) ≥40 kg/m2. All patients had normal intraoperative gallbladder sonography. Transabdominal sonography was obtained at 2, 4, and 6 months following surgery, or until gallstone formation. Results Of 233 patients with at least one postoperative sonogram, 56 were randomized to placebo, 53 to 300 mg ursodiol, 61 to 600 mg ursodiol, and 63 to 1,200 mg ursodiol. Preoperative age, sex, race, weight, BMI, and postoperative weight loss were not significantly different between groups. Gallstone formation occurred at 6 months in 32%, 13%, 2%, and 6% of the patients on the respective doses. Gallstones were significantly (P Conclusion A daily dose of 600 mg ursodiol is effective prophylaxis for gallstone formation following GBP-induced rapid weight loss.

Interleukin-4 is required for the induction of lung Th2 mucosal immunity.

Abstract: Aerosol antigen challenge of ovalbumin-sensitized mice induced an eosinophilic airway inflammation that was dependent on interleukin (IL)-5 and CD4+, but not CD8+, T lymphocytes. The involvement of the Th2 phenotype of CD4+ T cells was supported by demonstrating that FACS-sorted purified lung T cells from sensitized, but not control, mice produced IL-4, IL-5, and IL-10 after activation of the CD3/TCR complex. To determine the role of IL-4 in this process, we used mice in which the gene for IL-4 was deleted by homologous recombination. Antigen challenge of IL-4 gene-targeted mice resulted in a marked attenuation of eosinophilic inflammation and IL-5 secretion. To more fully understand the time when IL-4 was involved, we administered a neutralizing anti-IL-4 antibody (11B11) either immediately before antigen challenge or during immunization. Inhibition of IL-4 before antigen challenge had little effect on antigen-induced eosinophil infiltration. However, when 11B11 was administered during immunization, there was a marked reduction in eosinophil infiltration. Cross-linking of the CD3/TCR complex of FACS-sorted lung T cells revealed that only when anti-IL-4 was administered during immunization was there an inhibition of T cell-derived IL-5 and IgE production. These results suggest that IL-4 is central both to the induction of a local Th2 response and to the development of eosinophilic inflammation of the lung. Moreover, we suggest a sequential involvement of IL-4 and IL-5, with IL-4 committing naive T cells to a Th2 phenotype which upon activation by aerosol provocation secrete IL-5, resulting in eosinophil accumulation.

Chemically regulatable and anti-pathogenic DNA sequences and uses thereof

Abstract: The present invention provides recombinant or chimaeric DNA molecules comprising plant SAR genes, wherein the wild-type genes corresponding to said SAR genes can be chemically induced in a plant in a protein-synthesis independent or in a protein-synthesis dependent manner. Methods for obtaining these DNA molecules are also provided. Additionally, the present invention provides chemically inducible wheat genes, Arabidopsis chitinase IV, Maize PR-1mz, and Maize thaumatin PR-5mz. The anti-pathogenic sequences according to the invention can be genetically engineered and transformed into plants to obtain transgenic plants with enhanced resistance to disease. In one embodiment of the present invention, the chemically regulatable DNA promoter sequence of the Arabidopsis Pr-1 gene is provided. Another aspect of the present invention is a method of improving protection of a plant against a pest comprising transgenically expressing in said plant two or more DNA molecules encoding anti-pathogenic proteins, wherein the transgenically expressed proteins exert a synergistic effect.

MF59 Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines

Abstract: Advances in recombinant DNA technology have made possible the advent of a new generation of safer, better-defined subunit vaccines. Because vaccines based on these weakly immunogenic antigens require an adjuvant for efficacy, we undertook the development of a safe and efficacious adjuvant suitable for widespread human administration. Vaccines formulated with aluminum salts (alum), the only adjuvant thus far utilized with vaccines approved in the United States for human administration, were necessarily adopted as a benchmark for the minimum acceptable activity of a new adjuvant. Our goal was to develop an adjuvant that significantly exceeded aluminum hydroxide in potency, while retaining equally low toxicity. By the early 1990s a wide variety of approaches to adjuvant development had been described (Allison and Byars, 1990; Edelman, 1980; Gregoriadis and Panagiotidi, 1989; Warren et al., 1986). Two major mechanisms of adjuvant activity have been repeatedly cited in this literature: the depot effect, whereby long-term release of antigen results in increased immune response; and coadministration of immunostimulators, which specifically activate portions of the immune system in, as yet, incompletely defined fashions. The prototypic strong adjuvant, complete Freund’s adjuvant (CFA), combined these functions by releasing a mixture of immunostimulatory mycobacterial cell wall components along with antigen from a water/mineral oil/Arlacel A emulsion depot over an extended period of time (Freund, 1956). CFA remains the reference standard for potent adjuvant activity; however, it is now considered too toxic in many cases for use even in laboratory animals. In addition to the aluminum salts, several adjuvants based on the depot effect alone have been studied. These include incomplete Freund’s adjuvant (IFA), which lacks the potent, but toxic, cell wall components, and Adjuvant 65 (water/peanut oil/mannide monooleate), a yet further detoxified water/oil formulation (Hilleman et al., 1972a,b). Despite extensive study, neither formulation was approved for human administration. We chose to avoid water/oil emulsions. A more recent version of the depot approach, controlled release of antigen from synthetic polymer microspheres, remains a promising area of study (Cohen et al., 1991; O’Hagan et al., 1991) but appears to have an unacceptably long development time for our purposes.

Different susceptibility of protein kinases to staurosporine inhibition Kinetic studies and molecular bases for the resistance of protein kinase CK2

Abstract: A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen-activated protein (MAP) kinase and protein-tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK-1), c-Fgr, Lyn, CSK and TPK-IIB/p38Syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr-specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub-families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Met120 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glu170, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10- 100-fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which His160 has been replaced by Asp. It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

Abstract: The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5

Abstract: Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Transcriptional activation of the nuclear receptor RZR alpha by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand.

Abstract: Many important physiological functions are controlled by hormones via binding and activating members of the nuclear receptor superfamily. This group of structurally related transcription factors also includes a still growing number of orphan receptors for which no ligand is known so far. The identification of ligands for orphan receptors is a key to understanding their physiological role, as has been successfully shown for retinoid X receptors and the discovery of 9-cis retinoic acid as a specific ligand. We have discovered very recently that the pineal gland hormone melatonin is a specific ligand for the brain-specific nuclear receptor RZR beta. Here we report that the alpha-subtype of RZR, RZR alpha and its splicing variant ROR alpha 1, is also a nuclear receptor for melatonin with binding specificities in the low nanomolar range. In contrast to RZR beta, RZR/ROR alpha is expressed in many tissues and cells outside the brain. We found that RZR alpha and ROR alpha 1 vary in their constitutive transactivational activity and are activated to a different extent by melatonin. Furthermore, we identified a synthetic RZR-ligand, the thiazolidine dione CGP 52608. This compound is a functional analogue of melatonin at its nuclear receptor, but does not bind to the high affinity membrane receptor for melatonin. Therefore, this specific RZR-ligand may help to differentiate between nuclear and membrane signalling of melatonin.

Virus-specific CD8+ cells can switch to interleukin 5 production and induce airway eosinophilia.

Abstract: Virus infections of the lung are thought to predispose individuals to asthma, a disease characterized by eosinophil infiltration of the airways. CD8+ T cells are an important part of the host response to virus infection, however, they have no reported role in eosinophil recruitment. We developed a mouse model of virus peptide-stimulated CD8+ T cell immune responses in the lung. We found that bystander CD4+ T helper cell type 2 immune responses to ovalbumin switched the virus peptide-specific CD8+ T cells in the lung to interleukin (IL) 5 production. Furthermore, when such IL-5-producing CD8 T cells were challenged via the airways with virus peptide, a significant eosinophil infiltration was induced. In vitro studies indicated that IL-4 could switch the virus-specific CD8+ T cells to IL-5 production. These results could explain the link between virus infection and acute exacerbation of asthma and, perhaps more importantly, they indicate an IL-4-dependent mechanism that would impair CD8+ T cell responses and delay viral clearance from the host.

Phosphinic acid analogues of GABA. 2. Selective, orally active GABAB antagonists.

Abstract: In 1987, 25 years after the synthesis of the potent and selective GABA B agonist baclofen (1), Kerr et al. 5 described the first GABA B antagonist phaclofen 2. However, phaclofen and structurally similar derivatives 3-5 did not cross the blood-brain barrier and hence were inactive in vivo as central nervous system agents. As a consequence, the therapeutic potential of GABA B antagonists remained unclear. In exploring GABA and baclofen derivatives by replacing the carboxylic acid residue with various phosphinic acid groups, we discovered more potent and water soluble GABA B antagonists. Electrophysiological experiments in vivo demonstrated that some of the new compounds were capable of penetrating the blood-brain barrier after oral administration. Neurotransmitter release experiments showed that they interacted with several presynaptic GABA B receptor subtypes, enhancing the release of GABA, glutamate, aspartate, and somatostatin. The new GABA B antagonists interacted also with postsynaptic GABA B receptors, as they blocked late inhibitory postsynaptic potentials. They facilitated the induction of long-term potentiation in vitro and in vivo, suggesting potential cognition enhancing effects. Fifteen compounds were investigated in various memory and learning paradigms in rodents. Although several compounds were found to be active, only 10 reversed the age-related deficits of old rats in a multiple-trial one-way active avoidance test after chronic treatment. The cognition facilitating effects of 10 were confirmed in learning experiments in Rhesus monkeys. The novel GABA B antagonists showed also protective effects in various animal models of absence epilepsy

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance

Abstract: Summary Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have cetelasa activity. Based on this finding elevated levels of hydrogen peroxide (H202} were postulated to act as a second messenger of SA in the SAR signal trensduction pathway. A series of experiments have been carried out to clarify the role of H202 in SARsignaling. No increase of H202 was found during the onset of SAR. Induction of the SAR gene, PR-1, by H202 and H20 zinducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H202 induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H202, a dose-dependent accumulation of total SA species was found, suggesting that H202 may induce PR-1 gene expression through SA accumulation. While the results do not support a role for HzO2 in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.

Genes for the synthesis of antipathogenic substances

Abstract: The present invention is directed to the production of an antipathogenic substance (APS) in a host via recombinant expression of the polypeptides needed to biologically synthesize the APS Genes encoding polypeptides necessary to produce particular antipathogenic substances are provided, along with methods for identifying and isolating genes needed to recombinantly biosynthesize any desired APS The cloned genes may be transformed and expressed in a desired host organisms to produce the APS according to the invention for a variety of purposes, including protecting the host from a pathogen, developing the host as a biocontrol agent, and producing large, uniform amounts of the APS

Phosphinic acid analogues of GABA. 1. New potent and selective GABAB agonists.

Abstract: The antispastic agent and muscle relaxant baclofen 1 is a potent and selective agonist for bicuculline-insensitive GABAB receptors. For many years efforts to obtain superior GABAB agonists were unsuccessful. We describe the syntheses and biological properties of two new series of GABAB agonists, the best compounds of which are more potent than baclofen in vitro and in vivo. They were obtained by replacing the carboxylic acid group of GABA or baclofen derivatives with either the phosphinic acid or the methylphosphinic acid residue. Surprisingly, ethyl- and higher alkylphosphinic acid derivatives of GABA yielded novel GABAB antagonists, which are described in part 2 of this series. Structure-activity relationships of the novel GABAB agonists are discussed with respect to their affinities to GABAB receptors as well as to their effects in many functional tests in vitro and in vivo providing new muscle relaxant drugs with significantly improved side effect profiles.

Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms

Abstract: The present invention provides novel eukaryotic DNA sequences coding for native protoporphyrinogen oxidase (protox) or modified forms of the enzyme which are herbicide tolerant Plants having altered protox activity which confers tolerance to herbicides are also provided These plants may be bred or engineered for resistance to protox inhibitors via mutation of the native protox gene to a resistant form or through increased levels of expression of the native protox gene, or they may be transformed with modified eukaryotic or prokaryotic protox coding sequences or wild type prokaryotic protox sequences which are herbicide tolerant Diagnostic and other uses for the novel eukaryotic protox sequence are also described Plant genes encoding wild-type and altered protox, purified plant protox, methods of isolating protox from plants, and methods of using protox-encoding genes are also disclosed