Charles Burghardt

Leptin Receptor Long-form Splice-variant Protein Expression in Neuron Cell Bodies of the Brain and Co-localization with Neuropeptide Y mRNA in the Arcuate Nucleus

Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 956-1102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight.

Interaction of Phosphorylated FcϵRIγ Immunoglobulin Receptor Tyrosine Activation Motif-based Peptides with Dual and Single SH2 Domains of p72syk ASSESSMENT OF BINDING PARAMETERS AND REAL TIME BINDING KINETICS

To examine the characteristics of the interaction of the FcεRIγ ITAM with the SH2 domains of p72syk, the binding of an 125I-labeled dual phosphorylated FcεRIγ ITAM-based peptide to the p72syk SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72syk SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcεRIγ ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

The dopamine receptor of the rat mammotroph in cell culture as a model for drug action

Abstract Dopamine can act directly on pituitary cells to inhibit prolactin release. This action can be blocked by dopamine receptor blocking drugs such as haloperidol, sulpiride and other neuroleptic agents. Comparison of the properties of the mammotroph dopamine receptor with the adenylate cyclase linked dopamine receptor of the limbic forebrain reveals some obvious differences. For example, dopamine receptor stimulants such as S-584 and lergotrile mesylate are inactive in stimulating the adenylate cyclase preparations but are potent in inhibiting pituitary prolactin secretion. Such inhibition of prolactin secretion can be reversed by haloperidol or sulpiride. In contrast to these observations, sulpiride does not block dopamine stimulation of cAMP formation. In addition, dopamine, apomorphine or lergotrile mesylate have no effect on a pituitary adenylate cyclase preparation and dopamine fails to elevate cAMP in the intact cells in culture. Despite the similarity between these two dopamine sensitive systems with respect to a number of agonists and antagonists, the exceptions described suggest that the pituitary system with further study may offer some greater reliability as a predictive test for clinically useful agents. These results also suggest that the receptors for dopamine, like that for norepinephrine, are of two types, only one of which is coupled to adenylate cyclase.

Solid-phase extraction on silica cartridges as an aid to platelet-activating factor enrichment and analysis

Solid-phase extraction methods using pre-packed silica cartridges and various elution solvents have been developed and evaluated as chromatographic means to enrich biological lipid extracts for platelet-activating factor (PAF). The optimized procedure advanced selectively removed the major tissue/blood neutral lipids and non-choline-containing phospholipids from complex lipid mixtures and yielded thereby a choline phospholipid fraction markedly enriched in bioactive PAF. Some tested solid-phase extraction procedures, while capable of resolving choline phospholipids from other polar and non-polar species, were detrimental to PAFs bioactivity and evidenced considerable loss or degradation of this analyte. It is concluded that, with solvents of appropriate composition, strength and polarity, solid-phase extraction on silica cartridges has several unique advantages over conventional thin-layer and column chromatographic methods presently in use for PAF enrichment from biological sources.

Specific binding of 1- O -alkyl-2-acetyl- sn -glycero-3-phosphocholine (platelet-activating factor) to the intact canine platelet

Binding of 3H-labeled 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor; PAF) to the intact, washed canine platelet has been defined and characterized as being specific and receptor-mediated. Under the conditions described, specific binding to 2 X 10(7) canine platelets reached saturation within 10 min at a [3H]PAF concentration of approximately 0.4 nM. Non-specific binding was accountable for, at most, some 30% of the total PAF bound at equilibrium. Above approximately 0.4 nM [3H]PAF, total binding and non-specific binding increased in parallel. Since no involvement of PAF ligand in dog platelet intermediary metabolism during the binding incubation could be demonstrated, non-specific PAF binding may reflect a partitioning of the molecule into a cellular compartment (perhaps the platelet membranes). Equilibrium analysis revealed that the canine platelet has one class of specific binding sites with a Kd of 0.63 +/- 0.02 nM PAF, a Bmax of 222 +/- 10 fmol/10(7) platelets, and, at most, 1.33 +/- 0.06 X 10(3) binding sites/platelet. [3H]PAF specific binding to the canine platelet is ligand-selective and stereo-selective, as demonstrated by the relative abilities of non-labeled PAF and various PAF analogs/metabolites to inhibit [3H]PAF specific binding in a concentration-dependent manner. The extents to which PAF and PAF analogs were able to displace specifically-bound [3H]PAF from the canine platelet correlated well with their physiological (i. e., pro-aggregatory) effects. These data offer the first quantitative description of canine platelet high-affinity PAF binding sites/receptors and link receptor-mediated PAF binding to canine platelet physiology.

Radioligand competitive binding methodology for the evaluation of platelet-activating factor (PAF) and PAF-receptor antagonism using intact canine platelets

High-affinity, stereoselective, and ligand-selective specific binding of 3H-labeled platelet-activating factor (PAF) to its receptor on the dog platelet is reproducible over wide mass and concentration ranges of [3H]PAF. The [3H]PAF specific binding can be competitively inhibited by low picogram amounts of nonlabeled PAF. These observations have led to the formulation of radioligand competitive binding methodology for the detection and estimation of PAF in a biological lipid sample and the quantitative evaluation of PAF-receptor antagonism. The methodology is predicated upon correlation between the ability of a PAF analog/biological lipid sample/(synthetic) substance to inhibit [3H]PAF specific binding to the washed canine platelet and the known inhibition of [3H]PAF specific binding by standard, nonradioactive PAF. Application of this methodology to lipid extracts of human saliva has uncovered the finding that subjects with upper respiratory infection and chronic allergies have high saliva PAF contents. Pharmacologically active antiallergy agents known to inhibit PAF-induced pathology in animal models of disease were demonstrated, with the methology advanced, to act as PAF-receptor antagonists, and their potencies were quantified. These investigations indicate that the system proposed, in its ease, economy, sensitivity, specificity and capacity, has practical value for detecting and estimating PAF in biological lipid extracts and for evaluating PAF-receptor antagonism.

Effect of drugs on the hemolysis of rat erythrocytes.

Abstract Twenty-two well known drugs, covering a wide range of pharmacological types, have been examined for their ability to affect the hemolysis of rat erythrocytes induced by hypotonicity, deoxycholate and digitonin. Chlorpromazine, chlorprothixene, phenindamine, desipramine, imipramine and chloroquine enhanced deoxycholate-induced hemolysis and depressed hypotonie hemolysis at low concentrations while stimulating it at high concentrations. Debrisoquin, guanethidine, tripelennamine, leverphanol and cocaine, protected against hypotonie and enhanced deoxycholateinduced hemolysis. Acetylsalicylic acid, indomethacin, chlordiazepoxide, cortisol, cortisone, deoxycorticosterone, hydrochlorothiazide, acetazolamide and prostigmine protected against hypotonie hemolysis without affecting other systems. Digitonininduced hemolysis was least affected by these drugs. Ethacrynic acid was unique, even among other diuretics, ir protecting against deoxycholate- and digitonin-induced hemolysis. Ouabain was also unique in its complete inactivity in the three hemolytic systems. No correlation could be found between the pharmacological and membrane effects.

Discovery of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors

Through high throughput screening and subsequent hit identification and optimization, we synthesized a series of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as the first reported potent and reversible GFAT inhibitors. SAR studies of this class of compounds indicated significant impact on GFAT inhibition potency by substitutions on the A-ring and C-ring. The ketone group was found to be necessary for high potency. Compound 28 (RO0509347) demonstrated potent GFAT inhibition (IC(50)=1μM) with a desirable pharmacokinetic profile in rats, and showed significant efficacy in reducing the glucose excursion in an OGTT test in ob/ob mice.

Emotional Arousal and Platelet Physiology: A Review and Some Original Contributions

Abstract In our laboratory we have been studying the alterations in platelet physiology before, during and after emotional arousal. The findings indicate a clear relationship between affective states and the ability of platelets to respond to stimuli in vitro. The purpose of this article is both to review previous works in which the platelet was studied vis a vis the impact of emotional arousal and to present our new data which further support the interrelationship between platelet physiology and emotions.