Measurement of cerebral monoamine oxidase B activity using L-[11C]deprenyl and dynamic positron emission tomography

A tracer kinetic procedure was developed for the measurement of monoamine oxidase type B (MAO-B) activity using L-[11C]deprenyl and positron emission tomography (PET). The kinetic model consisted of two tissue compartments with irreversible binding to the second compartment (three rate constants). In addition, a blood volume component was included. Special attention was given to the accurate measurement of the plasma and whole blood input functions. The method was applied to the measurement of the dose-response curve of a reversible MAO-B inhibitor (Ro 19-6327). From the results, it followed that the rate constant for irreversible binding (k3) appeared to be a better index of MAO-B activity than the net influx constant Ki. Furthermore, regional analysis demonstrated that Ki, but not k3, was flow dependent. This implies that full kinetic analysis is required for an accurate assessment of MAO-B activity.     

Formation of free choline in brain tissue during in vitro energy deprivation

Free choline and ATP contents were measured in Mongolian gerbil hippocampal slices (tissue) and incubation media (media) during exposure to 30 min of aglycemia, high potassium, anoxia, or ischemia. Changes in choline levels reflected the degree of energy reduction, lower ATP levels being associated with high choline (4-fold increase during exposure to high potassium and anoxia, and 11-fold increase during ischemia). Media (extracellular) choline was particularly affected and increased about twofold during relatively mild energy depletion (e.g., aglycemia), but tissue choline content was less sensitive to energy reduction. A plot of choline vs. ATP levels indicated a nonlinear correlation, and the sharp increase in choline occurred when ATP values fell to about 2.5 nmol/mg of protein. Inhibition of acetylcholine sterase by 10 microM physostigmine during ischemia did not prevent an increase in choline contents but rather enhanced them, indicating that acetylcholine hydrolysis was not the source of free choline. Formation of free choline was Ca2+ independent. These findings suggest the involvement of phospholipase D and phosphatidylcholine hydrolysis in free choline formation during energy stress. The extent of choline formation may be an indicator of the degree of membranal damage, which in turn reflects damage to the metabolic machinery of the cell.     

Time-dependent effects of ovarian steroids on tyrosine hydroxylase activity in the limbic forebrain of female rats

Summary In this work, we have studied the time-course of the effects of pharmacological administration of ovarian steroids on tyrosine hydroxylase (TH) activity in the limbic forebrain of ovariectomized rats. Administration of estradiol produced a late decrease in TH activity. This effect was found 24 hours after the last steroid injection, disappearing at 32 hours. It was antagonized by progesterone, since a single injection of this steroid to estradiol-pretreated rats reversed to control values the estradiol-induced decrease. Nevertheless, the administration of progesterone after estradiol treatment caused a short-time decrease in the limbic activity of TH, which was observed 4 hours after the last steroid injection, disappearing subsequently. On the other hand, the administration of progesterone alone produced a biphasic effect, with a reduction at 24 hours, followed by an increase at 32 hours. These effects were only observed in the animals non-treated with estradiol, disappearing with a previous treatment with estrogens. Hence, it can be concluded that both ovarian steroids may affect the limbic TH activity. Thus, estradiol produced a late inhibitory effect on the activity of this enzyme, which was antagonized by progesterone. Administration of the last one to estradiol-treated rats produced a short-time inhibitory effect, whereas its administration to non-treated rats produced a late biphasic effect (inhibition followed by stimulation), which was not observed in estradiol-treated rats.     

Ubiquitin and heat shock protein expression in amyotrophic lateral sclerosis

The expression of two heat shock proteins, HSP72 and p57, in addition to ubiquitin, has been studied immunocytochemically in nine amyotrophic lateral sclerosis (ALS) cases and 10 age-matched controls. HSP72 and p57 antibodies did not identify the characteristic ubiquitin-immunoreactive inclusions present in anterior horn cells in ALS spinal cord. Antibodies to HSP72, but not to p57 or ubiquitin, strongly labelled structures corresponding to polyglucosan bodies in spinal grey matter. Such immunoreactive profiles were more abundant in ALS cases, although they were also present in control material. They were sometimes identified by haematoxylin and eosin and periodic acid Schiff reaction, but were not labeled by phosphotungstic acid haematoxylin or by antibodies to glial fibrillary acidic protein. Although ubiquitin, HSP72 and p57 are stress-induced proteins, they are expressed differently and might therefore have different significance in neuronal degeneration.     

Improved adhesion and proliferation of human endothelial cells on polyethylene precoated with monoclonal antibodies directed against cell membrane antigens and extracellular matrix proteins.

Endothelial cell seeding may improve the patency of synthetic vascular grafts provided that platelet reactivity of nonendothelialized sites is not increased. We have investigated if surface-adsorbed monoclonal antibodies directed against endothelial cell membrane proteins and against extracellular matrix proteins promote the adhesion and proliferation of cultured human endothelial cells, without causing platelet deposition at non-endothelialized sites. Adhesion of endothelial cells onto polyethylene coated with monoclonal antibodies directed against endothelial cell-specific membrane antigens, integrin receptors and glycoprotein CD31 was equal to or higher than adhesion onto fibronectin-coated polyethylene. Endothelial cells did not proliferate on these surface-adsorbed antibodies. However, pre-coating of polyethylene with mixtures of endothelial cell-specific monoclonal antibodies and monoclonal antibodies directed against fibronectin or von Willebrand factor, resulted in relatively high adhesion and optimal proliferation. Platelet reactivity of the polyethylene surface was found to significantly increase after adsorption of fibronectin, endothelial cell-specific monoclonal antibody or its Fc fragments. In contrast, adsorption of F(ab')2 fragments of endothelial cell-specific monoclonal antibody did not promote platelet deposition. Therefore, it is concluded that coating of vascular graft materials with mixtures of F(ab')2 fragments of monoclonal antibodies specifically directed against endothelial cells and against extracellular matrix proteins may be an effective way to both promote the growth of seeded endothelial cells and limit platelet-graft interaction.     

The use of laser-light scattering and controlled shear in platelet aggregometry

Laser-light scattering was used to observe and quantify the dynamics of human blood platelet aggregation in platelet-rich plasma (PRP). Aggregation was performed in a controlled shear environment by placing the PRP in the annular space between a rotating cylindrical rod and a stationary cylindrical tube. The instrument was capable of very sensitive continuous semi-quantitative measurements of chemically-induced microaggregation. As a demonstration of the technique, results are presented for ADP-induced aggregation at doses of 10, 1, and 0.1 microM and collagen-induced aggregation at a dose of 5 micrograms/ml, each at shear rates of 1,000 s-1 and 500 s-1. Extensive aggregation was observed in response to ADP at even the low dose of 0.1 microM, indicating a high sensitivity to microaggregates. The sensitivity of the ultimate size of the ADP-induced aggregates to ADP concentration was shear dependent. The formation of microaggregates by collagen stimulation was shown to be almost immediate, as contrasted with a 10-20 s typical lag when observed turbidometrically. Disaggregation was observed with 1 microM ADP, but this was only partial, as contrasted with the complete recovery of transmittance observed in the turbidometric technique. Electronic particle sizing and counting was employed to semiquantitatively verify the aggregate size distributions found from mathematical conversion of the laser-light scattering data.     

Dopaminergic transmission in the hypothalamic arcuate nucleus to produce acupuncture analgesia in correlation with the pituitary gland

Acupuncture analgesia (AA) caused by low frequency stimulation of the acupuncture point (AP) was abolished by hypophysectomy and adrenalectomy. Termination of the AA producing pathway from the AP to the pituitary gland was in the medial hypothalamic arcuate nucleus (M-HARN). The origin of the descending pain inhibitory system associated with AA was in the posterior HARN (P-HARN). AA in the hypophysectomized rats, and enhanced neuronal activity in the P-HARN that were abolished during acupuncture stimulation, were both restored by intraperitoneal microinjection of 0.5 mg/kg morphine or 0.1 micrograms beta-endorphin into the P-HARN during acupuncture stimulation. Of the analgesia produced by dopamine or beta-endorphin injected into the P-HARN, that caused by beta-endorphin disappeared after denervation of the M-HARN. The P-HARN neurons that responded to acupuncture stimulation also responded to iontophoretic dopamine, but not to iontophoretic morphine nor ultramicroinjected beta-endorphin. The transmission between the M-HARN and P-HARN may be dopaminergic, and beta-endorphin might presynaptically modulate this transmission. Reduction of sodium ions may have been the reason for abolition of AA after adrenalectomy.     

Antibodies to interleukin-1 inhibit cytokine-induced proliferation of neonatal rat Schwann cells in vitro

Unfractionated cytokines have been shown to induce in vitro proliferation of neonatal rat Schwann cells but the nature of the mitogen(s) is not known. A mixture of rabbit antibodies specific for recombinant interleukin-1α (IL-1α) and interleukin-1β (IL-1β) inhibited Schwann cell proliferation induced by unfractionated human cytokines whereas antibodies to interleukin-2 (IL-2) and control IgG did not. However, purified human IL-1 and recombinant human IL-1α or β did not induce Schwann cell proliferation on their own.