A simple, sensitive and reliable assay for serotonin and 5-HIAA in brain tissue using liquid chromatography with electrochemical detection

An extremely sensitive and simple method for simultaneously measuring both serotonin and 5-hydroxyindoleacetic acid (5-HIAA) has been developed using liquid chromatography (LC) and electrochemical detection. Assay conditions are described which resolve and measure as little as 22 picograms of serotonin and its deaminated metabolite in deproteinated brain samples.     

Odor psychophysics and sensory engineering

This paper illustrates the utilization of psychophysical scalingand optimization technology to accomplish two goals. First,by means of ‘goal programming’ the odor scientistcan interrelate chemicals and odor qualities, specify a desiredprofile of odor qualities and determine that combination ofodorants which come as close as possible to generating thatdesired profile. Second, by means of non-linear optimization,the scientist can determine the relation between chemical concentrationsand liking, and furthermore ascertain that combination of chemicalconcentrations which generate maximal liking ratings, subjectto constraints. Constraints include chemical concentrationslying within prespecified limits, and sensory perceptions lyingwithin specific bounds. Mixtures of butyric acid and pulegoneillustrate the approach.     

Immunoglobulin G-induced experimental chronic immune synovitis: cell-mediated immunity to native interstitial collagen molecules and their constituent polypeptide chains

In vitro cell-mediated immune responses to homologous rabbit immunoglobulin G (IgG), purified protein derivative (PPD), native Type I, II, and III collagen, and denatured Type I, II, and III collagen were studied in an IgG-induced animal model of immune synovitis. Immune response was measured as augmented [3H]thymidine incorporation by spleen cells on exposure to antigen. Immune responses were observed in vitro after 72 hr of culture with antigen, while a majority of responses to antigens occurred after 96 hr of incubation. Separation of spleen cell subpopulations showed that measured immune responses were of T-cell origin. In vitro cell-mediated immune responses were observed for native and denatured collagen in splenic cell cultures from six of seven synovitic rabbits (P less than 0.01) but not in control spleen cell cultures derived from normal, adjuvant-primed or IgG-immune nonsynovitic rabbits. The incidence of cellular reactivity to incubation with native interstitial collagens was as follows: Type I, 43%; Type II, 43%; Type III, 57%. The incidence of in vitro immune responses to denatured collagens in cultures derived from rabbits with synovitis was: Type I, 50%; Type II, 50%; Type III, 67%. The relatively high incidence of immune response to both native and denatured collagens suggests that immunity to structural components of the synovial membrane and the adjacent surface of articular cartilage may play a role in the inflammation observed in immune synovitis.     

THE PERMISSIVE ROLE OF HYPERTHERMIA IN THE DISAGGREGATION OF BRAIN POLYSOMES BY l-DOPA OR d-AMPHETAMINE1

Abstract— l -DOPA or d -amphetamine administration disaggregates brain polyribosomes in animals maintained in an environment warm enough (26°C) so that the drugs concurrently elevate their body temperatures to above 39°C. The production of equivalent hyperthermia (by keeping control rats at ambient temperatures of 40–44° C) does not cause similar disaggregation of brain polysomes. Hence, the role of hyperthermia in the drug-induced disaggregation is permissive.     

Attenuation by catecholamine antagonists of the hypothermia that follows cerebral infarction in gerbils.

The hypothermia that follows induction of cerebral infraction in gerbils can be attenuated by drugs which reduce the interactions between endogenous brain catecholamines and postsynaptic receptors. This observation is compatible with the hypothesis that stroke-induced hypothermia is mediated, in part, by the release of catecholamines from ischemic neurons.     

Silver Protein Staining of Nerve Fibers in Celloidin Sections

Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr at 37 C, and transferred to 1% concentrated NH₄OH in 50% alcohol 15-18 hr at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 M H₃BO₃/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 gm of copper foil, 0.002 inch thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO₃, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr at 37 C, rinsed in distilled water, reduced in a solution of 5% Na₂SO₃ and 1% hydroquinone for 10 min, washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec, washed in distilled water and placed in 5% Na₂S₂O₃ for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.     

The Effect of Molecular Weight,PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)₂ (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)₂versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)₂ with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.     

Selective inactivation or reconstitution of adenosine A2A receptors in bone marrow cells reveals their significant contribution to the development of ischemic brain injury

Inactivation of the adenosine A(2A) receptor (A(2A)R) consistently protects against ischemic brain injury and other neural insults, but the relative contribution of A(2A)Rs on peripheral inflammatory cells versus A(2A)Rs expressed on neurons and glia is unknown. We created a chimeric mouse model in which A(2A)Rs on bone marrow-derived cells (BMDCs) were selectively inactivated or reconstituted by bone marrow transplantation. Selective reconstitution of A(2A)Rs on BMDCs (A(2A)R knockout mice transplanted with wild-type bone marrow cells) largely reinstates ischemic brain injury in global A(2A)R knockout mice. Conversely, selective inactivation of A(2A)Rs on BMDCs (wild-type mice transplanted with A(2A)R knockout bone marrow cells) attenuates infarct volumes and ischemia-induced expression of several proinflammatory cytokines in the brain, but exacerbates ischemic liver injury. These results indicate that the A(2A)R-stimulated cascade in BMDCs is an important modulator of ischemic brain injury and that ischemic brain and liver injuries are regulated distinctly by A(2A)Rs on BMDCs.