Transient neuritogenesis in NB2a/d1 neuroblastoma cells induced by glial-derived protease inhibitors

The initial outgrowth of neuritogenesis in mouse NB2a/d1 neuroblastoma cells may be regulated by thrombin or a thrombin-like protease, present either in serum or adsorbed to the plasma membrane, since neuritogenesis is induced by serum deprivation and treatment with the specific thrombin inhibitor, hirudin (Shea et al., 1991, J. Neurochem., 56:842). Cultured astroglial cells secrete factors that promote neuritogenesis, including protease inhibitors active against thrombin, leading to suggestions that the inhibition of specific neuronal surface proteases by the surrounding glial environment may represent an initial step in axonal outgrowth in situ. To examine the relative importance of glial-derived protease inhibitory activities on neurine outgrowth, we tested the neurite promoting effect of glial-conditioned medium (GCM) on NB2a/d1 cells. Like serum deprivation and hirudin treatment, GCM induced neurite outgrowth within 4 hr. Exogenous thrombin inhibited the effect of GCM, and cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM, suggesting that GCM induces neuritogenesis by inhibition of a thrombin-like protease. Unlike neurites induced by serum removal or hirudin addition, which are rapidly resorbed following serum replenishment or hirudin depletion, however, GCM-induced neurites continued to elongate after GCM removal. Furthermore, cultures treated simultaneously with GCM and thrombin exhibited delayed outgrowth of neurites following GCM removal which were insensitive to further thrombin treatment. These findings indicate that the initial elaboration of neurites can be mediated by glial-derived protease inhibitor(s) active against a thrombin-like protease, but indicate the requirement of additional glial-derived factors for the maintenance and continued elaboration of these neurites.     

Effects of cholinergic muscarinic agents on protein kinase C activity in rat pineal gland.

The role of protein kinase C (PKC) on muscarinic regulation of serotonin release in the pineal gland was investigated by measuring the pineal-PKC activity and serotonin secretion in response to muscarinic agents. Pineal slices, short-term incubated (0-15 min) without additions produced a low serotonin release and 20 to 24 percent PKC activity was found associated with membrane fractions. Prolonged exposure of pineal slices (30-180 min) produced further translocation of PKC activity to the membranes and a significant increase of serotonin release. Short-term treatment with pilocarpine and carbachol, stimulated PKC activity of both cytosolic and particulate fractions and the release of pineal serotonin. The pilocarpine effect was blocked by atropine indicating that it was mediated by muscarinic receptors. The present data support that PKC activation correlates with the increase of serotonin release by muscarinic agonist in pineal gland.     

Brain metabolic activity associated with long-term memory consolidation

The use of day-old chickens trained on a single-trial passive avoidance task provides a useful paradigm for investigations into cellular mechanisms underlying memory formation. Pharmacological intervention studies indicate that there are three temporally identifiable stages of memory processing leading to the consolidation of information for this task. These stages, designated as short-term (STM; up to 15 min), intermediate-term (ITM; 15-55 min), and long-term (LTM; more than 55 min) memory, have been found to be sequentially dependent (Ng and Gibbs, 1989). In addition, ITM appears to consist of two physiologically distinguishable phases, A and B. Evidence in this laboratory suggests that the crossover between these ITM phases (at 30 min after training) represents a critical time-point for the triggering of LTM.     

Electrostatic interaction between ion-penetrable multi-layered membranes

A theory of the double layer interaction regulated by the Donnan potential between two ion-penetrable membranes in an electrolyte solution developed previously by Ohshima and Kondo is extended to the case in which the membranes consist of many layers having different thickness and densities of membrane-fixed charges. The interaction force is found to be determined mainly by the contributions from layers located within the depth of 1/kappa (kappa, Debye-Hückel parameter) from the membrane surface. It is also predicted that the interaction force may alter its sign with changing electrolyte concentration.     

A follow-up study of 85 patients with Graves' disease in remission who developed undetectable serum thyroid-stimulating hormone concentrations using sensitive TSH assays.

Eighty-five patients with Graves' disease in clinical remission after treatment for over 1 year by methimazole therapy (36 patients, group A) or subtotal thyroidectomy (49 patients, group B) who became undetectable for serum thyrotropin levels (TSH less than 0.05 mU/l), were further followed for 1 year or more. Eight patients in group A (22%) and 7 patients in group B (14%) relapsed. Eleven patients in group A (30%) and 5 patients in group B (10%) had fluctuating patterns of free T4 in the upper normal to slightly supranormal range indicative of subclinical hyperthyroidism. The remaining patients continued to have undetectable TSH levels or restored normal TSH levels and normal thyroid hormone concentrations in sera. The results of the present study indicate that the occurrence of undetectable serum TSH concentrations in Graves' disease patients previously treated with methimazole or surgery are not necessarily predictive of clinical relapse because the eventual outcome is variable.     

Syntheses, structures and anorectic effects of human and rat amylin.

Amylin, a 37-residue polypeptide with a single disulfide bond originally isolated from the pancreas of type-II diabetic patients, has been shown to cause peripheral insulin resistance and to attenuate the inhibition of hepatic glucose output by insulin. We have also shown that amylin is present in the rat hypothalamus and that it inhibits food intake by rats. In order to further investigate the anorectic properties we synthesized both human and rat amylin by the solid phase method and purified to homogeneity in an overall yield of 10-20%. Structural analyses indicated that human amylin exhibited predominantly a beta-sheet structure at both acidic and alkaline pH, whereas no ordered structure was evident in the case of rat amylin. Intrahypothalamic injection of rat amylin resulted in a potent dose-dependent inhibitory effect on the food intake by rats adapted to eat their daily ration of food in an eight-hour period. Human amylin was less effective as an anorectic agent. Furthermore, rat amylin completely blocked the potent orexigenic effect of neuropeptide Y (NPY). These investigations show that there is a fundamental difference in the secondary structures of human and rat amylin and that rat amylin is a potent inhibitor of both basal and NPY-induced feeding by rats.     

Linkage of bovine erythrocyte antigen loci B, C, L, S, Z, R' and T' and the serum protein loci post-transferrin 2 (PTF 2), vitamin D binding protein (GC) and albumin (ALB) to DNA microsatellite markers

Seven bovine erythrocyte antigen loci and three serum protein loci were tentatively assigned to chromosomes or synteny groups by linkage analysis to previously assigned microsatellite DNA markers. The erythrocyte antigen locus EAB was mapped to synteny group U27; EAC to chromosome 18, synteny group U9; EAL to chromosome 3, synteny group U6; EAS to chromosome 21, synteny group U4; EAZ to chromosome 10, synteny group U5; EAR' to chromosome 16, synteny group U1; and EAT' to chromosome 19, synteny group U21. The vitamin D binding protein (GC) and albumin (ALB) loci were assigned to chromosome 6, synteny group U15 and post-transferrin 2 (PTF 2) to chromosome 19, synteny group U21.     

Antiobesity and Antidiabetic β-agonists: Lessons Learned and Questions to be Answered

In several species of obese animals, a group of phenethanolamine β-agonists stimulates lipolysis and thermogenesis, resulting in the loss of body fat and weight. Brown adipose tissue is considered to be the major target tissue for the antiobesity activity of these compounds. Independent of this antiobesity activity, some of these compounds are also antidiabetic and increase muscle mass. Based on the pharmacological profile of these com-pounds, a npeceptor was proposed and character-ized in mouse, rat, and humans. The 133-receptor in brown adipose tissue has been suggested to mediate the antiobesity activity of these 13-agonists. Whether this receptor is responsible for the antidiabetic activ-ity and whether there is a linkage between the antiobesity/antidiabetic activity and the nutrient par-titioning activity is not clear. Clinical trials with these mixed 13-agonists showed marginal antiobesity effects when caloric intake of subjects was restricted. Insulin sensitivity was also improved in some of the trials designed to test the antidiabetic activity of these compounds. Side effects included tachycardia and tremor. To eliminate these side effects, a second generation of compounds was selected for its agonist activity on rat D3-receptors. Clinical trials with these compounds have shown lit-tle increase of energy expenditure even at high doses. Successful development of an antiohesity and antidi-abetic drug from this class of compounds will require the elucidation of the physiological role of the human 133-receptor and the regulatory mecha-nism between fuel efficiency and feeding behavior.