Localization of the nerve growth factor receptor on fetal human schwann cells in culture

Previous studies have established that Schwann cells (SC) in culture express an NGF receptor. In this study, cultures of fetal human SC were established from fetal nerves and various light microscopic (LM) and electron microscopic (EM) techniques were used to localize the NGF receptor on the SC. Results indicate that NGF receptor is localized to the plasma membrane of the SC. Quantitative digital analysis determined that the distal portion of the SC process had high concentrations of NGF receptor. The possible functional significance of this latter observation is discussed in terms of SC migration and ensheathment of axons.     

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

The effect of modifications of the A5 and A19 amino acid residues on the biological activity of insulin. [Leu5-A] and [Phe19-A] sheep insulins

Two analogs of sheep insulin, both differing from the native material by a single amino acid in the A chain, have been synthesized and isolated in highly purified form by procedures developed in this laboratory. In one case, the glutamine residue in position A⁵ was replaced by leucine ([Leu⁵-A]); in the other, the tyrosine residue in position A¹⁹ was replaced by phenylalanine ([Phe¹⁹-A]). The biological behavior of these analogs was compared with natural bovine insulin inin vitro tests and in receptor-binding assays, as well as in radioimmunoassay. In the stimulation of glucose oxidation by rat adipocytes, the analogs gave relative potencies of 30% and 7.8% for [Leu⁵-A] and [Phe¹⁹-A], respectively. Receptor-binding assays in rat liver plasma membranes showed similar behavior for both analogs. In radioimmunoassay, [Leu⁵-A] displayed a relative potency of 27.9%, while [Phe¹⁹-A] showed a relative potency of 19–27%, compared with bovine insulin. At high concentration, both analogs displayed the same maximal activity as bovine insulin, and the dose-response curves are essentially parallel. It is speculated that the interaction between the glutamine residue in position 5 and the tyrosine residue in position 19 of the A chain of insulin are important in maintaining a three-dimensional structure commensurate with high biological activity. The full intrinsic activity of both analogs at high concentrations and the similarity of the potency figures in receptor-binding and glucose-oxidation assays permit the further conclusion that the reduced potency in the latter assay can be ascribed wholly to the reduced binding affinity toward insulin receptors caused by the substitutions made in the analogs. The receptor-analog complexes are fully capable of triggering the next event in the chain leading to the biological response.     

A Nitrogen Pressure-Shearing Apparatus for Preparing Distinct Populations of Chromatin Fractions

A method for preparing reproducible chromatin fractions is described. Fragmentation is accomplished by expelling whole chromatin from an enclosed stainless-steel chamber through a restricted orifice utilizing low nitrogen pressure. Sheared chromatin is fractionated into two distinct heavy and light fractions by centrifugation on 5–20% linear sucrose gradient.     

Roux-en-Y Gastric Bypass Increases Intravenous Ethanol Self-Administration in Dietary Obese Rats

Roux-en-Y gastric bypass surgery (RYGB) is an effective treatment for severe obesity. Clinical studies however have reported susceptibility to increased alcohol use after RYGB, and preclinical studies have shown increased alcohol intake in obese rats after RYGB. This could reflect a direct enhancement of alcohol’s rewarding effects in the brain or an indirect effect due to increased alcohol absorption after RGYB. To rule out the contribution that changes in alcohol absorption have on its rewarding effects, here we assessed the effects of RYGB on intravenously (IV) administered ethanol (1%). For this purpose, high fat (60% kcal from fat) diet-induced obese male Sprague Dawley rats were tested ∼2 months after RYGB or sham surgery (SHAM) using both fixed and progressive ratio schedules of reinforcement to evaluate if RGYB modified the reinforcing effects of IV ethanol. Compared to SHAM, RYGB rats made significantly more active spout responses to earn IV ethanol during the fixed ratio schedule, and achieved higher breakpoints during the progressive ratio schedule. Although additional studies are needed, our results provide preliminary evidence that RYGB increases the rewarding effects of alcohol independent of its effects on alcohol absorption.     

Rare causes of scoliosis and spine deformity: experience and particular features

Background

Spine deformity can be idiopathic (more than 80% of cases), neuromuscular, congenital or neurofibromatosis-related. However, there are many disorders that may also be involved. We present our experience treating patients with scoliosis or other spine deformities related to rare clinical entities.

Methods

A retrospective study of the records of a school-screening study in North-West Greece was performed, covering a 10-year period (1992–2002). The records were searched for patients with deformities related to rare disorders. These patients were reviewed as regards to characteristics of underlying disorder and spine deformity, treatment and results, complications, intraoperative and anaesthesiologic difficulties particular to each case.

Results

In 13 cases, the spine deformity presented in relation to rare disorders. The underlying disorder was rare neurological disease in 2 cases (Rett syndrome, progressive hemidystonia), muscular disorders (facioscapulohumeral muscular dystrophy, arthrogryposis) in 2 patients, osteogenesis imperfecta in 2 cases, Marfan syndrome, osteopetrosis tarda, spondyloepiphyseal dysplasia congenita, cleidocranial dysplasia and Noonan syndrome in 1 case each. In 2 cases scoliosis was related to other congenital anomalies (phocomelia, blindness). Nine of these patients were surgically treated. Surgery was avoided in 3 patients.

Conclusion

This study illustrates the fact that different disorders are related with curves with different characteristics, different accompanying problems and possible complications. Investigation and understanding of the underlying pathology is an essential part of the clinical evaluation and preoperative work-up, as clinical experience at any specific center is limited.     

Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. Here, we perform a theoretical study of a model for morphogen spreading based on extracellular diffusion, which takes into account receptor binding and trafficking. We compare profiles of ligand and surface receptors obtained in this model with experimental data. To this end, we monitored directly the pool of surface receptors and extracellular Dpp with specific antibodies. We conclude that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement.     

In situ localization of TNFalpha/beta,TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue

Tumor necrosis factor (TNF) has been implicated in several infectious and inflammatory lung diseases. Two closely related variants, TNFalpha and TNFbeta, elicit various cellular responses via two distinct TNF receptors, the 55-kDa TNF-R1 and the 75-kDa TNF-R2. Recently, a TNFalpha-converting enzyme (TACE) was described, which cleaves and releases the membrane-bound TNFalpha. In the present study in normal rat and human lung tissue, the constitutive expression of TNFalpha/beta, TACE and TNF-R1/R2 was investigated by immunohistochemical techniques. In addition, TNFalpha and TNFbeta mRNA were localized by in situ hybridization. Both TNFalpha and TNFbeta were detected in various lung cell types. Expression of TNFalpha was particularly prominent in bronchial epithelial cells and vascular smooth muscle cells, next to alveolar macrophages. Both in situ hybridization for TNFalpha message and TACE immunostaining matched this expression profile. TNFbeta-so far only known to be produced by lymphocytes-was demonstrated in alveolar macrophages, bronchial epithelial cells, vascular smooth muscle cells and endothelial cells at the protein and the message level. Both TNF receptors were detected, with TNF-R1 being prominent on bronchial epithelial cells and endothelial cells, and TNF-R2 being expressed by nearly all cell types. Following LPS stimulation in isolated rat lungs TNFalpha/beta signal intensity was largely reduced due to liberation of stored TNFalpha/beta, while TACE immunoreactivity remained unchanged or was enhanced, demonstrating increased TNF generation.We conclude that both TNFalpha and TNFbeta are constitutively expressed by several non-leukocytic cell types in the human and rat lung. In concert with the expression of TACE and the TNF receptors R1 and R2, this finding suggests in addition to the known role of the TNF system in inflammation physiological functions of the TNF system in different compartments of the adult lung, with the vasculature and the bronchial tissue being of particular interest in addition to the leukocyte/macrophage populations.     

The Nitric Oxide-Cyclic GMP Pathway Plays an Essential Role in Both Promoting Cell Survival of Cerebellar Granule Cells in Culture and Protecting the Cells Against Ethanol Neurotoxicity

Abstract: NMDA has two beneficial effects on primary neuronal cultures of cerebellar granule cells (CGCs) established from 10-day-old rat pups. First, NMDA is neurotrophic and will enhance survival of CGCs in culture in the absence of ethanol. Second, ethanol exposure will induce cell death in CGC cultures, and NMDA can lessen this ethanol-induced cell loss, i.e., NMDA is neuroprotective. Because NMDA can stimulate production of nitric oxide (NO), which can in turn enhance synthesis of cyclic GMP, this study tested the hypothesis that the NO-cyclic GMP pathway is essential for NMDA-mediated neurotrophism and neuroprotection. Inhibiting the synthesis of NO with N ^G-nitro- l -arginine methyl ester eliminated both the NMDA-mediated neurotrophic and neuroprotective effects. Similarly, inhibiting production of cyclic GMP with the agent LY83583 also abolished these effects. The NO generator 2,2'-(hydroxynitrosohydrazono)bisethanamine produced neurotrophic and neuroprotective effects that were similar to those induced by NMDA. Also, 8-bromo-cyclic GMP produced neurotrophic and neuroprotective effects that were quite similar to the effects produced by NMDA. In conclusion, NMDA enhances survival of cerebellar granule cells and protects the cells against ethanol-induced cell death by a mechanism(s) that involves the NO-cyclic GMP pathway.