Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA

Abstract: GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

Summary Methods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide, Cu₂Fe(CN)₆·7 H₂O) at their subcellular sites. The small amounts of Hatchett's brown deposited as a result of the enzyme's activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchett's brown as a catalyst to effect the oxidative coupling of 3,3-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.Supported by U.S.P.H.S. Grant DE-02668.Dr. Anderson's work was performed at the Department of Anatomy of the University of Chicago and was supported by Research Grant No. M 71-077C from the Population Council.     

Community ecology of small mammal populations in Panamá following an outbreak of Hantavirus pulmonary syndrome.

In late 1999 and early 2000, an outbreak of hantavirus pulmonary syndrome (HPS) occurred in and around Los Santos, on the Azuero Peninsula of southwestern Panamá. This HPS episode, resulting in 22% case fatality, was linked to the Costa Rican pigmy rice rat, Oligoryzomys fulvescens costaricensis, which harbored a then undescribed hantavirus, Choclo virus. In addition, Cherrie's cane rat, Zygodontomys brevicauda cherriei, was identified as carrying a distinct hantavirus, Calabazo virus with no known pathogenicity to humans. Herein we present the ecological results of the outbreak investigations in the Azuero region. A total of 164 animals were captured, of which 126 were potential small, non-volant mammal hosts of a hantavirus: rodents in the family Muridae. There were significant differences in small mammal community structure between case sites and a negative control site. Differences were manifest in ecological measures of species diversity and in species evenness and heterogeneity measures, as indicated by Pairwise Euclidian distances and Morisita indices of community similarity. Our analyses suggest that human activities (i.e., deforestation for cattle ranching) coupled with environmental factors (i.e., increased precipitation) may have synergistically coalesced for an increased risk of HPS to area residents.     

Lead optimization of methionine aminopeptidase-2 (MetAP2) inhibitors containing sulfonamides of 5,6-disubstituted anthranilic acids

A series of aryl sulfonamides of 5,6-disubstituted anthranilic acids were identified as potent inhibitors of methionine aminopeptidase-2 (MetAP2). Small alkyl groups and 3-furyl were tolerated at the 5-position of anthranilic acid, while -OCH(3), CH(3), and Cl were found optimal for the 6-position. Placement of 2-aminoethoxy group at the 6-position enabled interaction with the second Mn(2+) but did not result in enhancement in potency. Introduction of a tertiary amino moiety at the ortho-position of the sulfonyl phenyl ring gave reduced protein binding and improved cellular activity, but led to lower oral bioavailability.     

Thienopyridine urea inhibitors of KDR kinase

A series of substituted thienopyridine ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR kinase activity. Several of these analogs, such as 2, are potent inhibitors of KDR (<10 nM) in both enzymatic and cellular assays. Further characterization of inhibitor 2 indicated that this analog possessed excellent in vivo potency (ED50 2.1 mg/kg) as measured in an estradiol-induced mouse uterine edema model.     

Patterns and drivers of stream benthic macroinvertebrate beta diversity in an agricultural landscape

Hydrobiologia - Laguna del Sauce, one of the main drinking water sources in Uruguay, is an eutrophic shallow lake with high temporal variation of inorganic turbidity caused by extreme wind events....     

Development of an LC–MS/MS assay to determine plasma pharmacokinetics of the radioprotectant octadecyl thiophosphate (OTP) in monkeys

Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD_80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid–liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1 → 95.0 and 403.1 → 95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.