Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor

Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride.     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Purification of proteins from polyacrylamide gels, free of detergent or dye

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.     

Three-dimensional organization and topographical features of the myenteric plexus (Auerbach) in the porcine small intestine: scanning electron microscopy after enzymatic digestion and HCl-hydrolysis

The present scanning electron microscopical (SEM) study was initiated to visualize the surface topography of Auerbach's plexus in the ileum of the pig. After enzymatic digestion of the connective-tissue components of the tunica muscularis and the tunica serosa followed by glutaraldehyde fixation, HCl-hydrolysis and stripping off either the longitudinal or circular smooth muscle layer, the three-dimensional architecture and topographical features of the myenteric plexus can clearly be observed. In this way, ganglia, primary, secondary and tertiary strands, and single nerve fibres can be demonstrated. Individual nerve cells, which are incompletely covered by glial cells and by remnants of the basal lamina, can be recognized in the centre and periphery of the ganglia and adjacent to primary and secondary nerve strands.     

Fine structural distinction between ganglia of the outer and inner submucosal plexus in porcine small intestine

Ultrastructural differences between ganglia of the plexus submucosus internus (Meissner; PSI) and plexus submucosus externus (Schabadasch; PSE) are described. Comparison revealed a different glia index (ratio glia per neuron) between the PSE (3:1) and the PSI (1:1), the arrangement of PSI neurons in compartments and the appearance of broad membrane-to-membrane appositions inside the compartments of the PSI. Structural and immunohistochemical differences between the two plexuses are discussed. In general, PSE neurons show a wider variety in size and shape than most of the PSI neurons.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

Distribution, morphology and projections of nitrergic and non-nitrergic submucosal neurons in the pig small intestine

 Sequential nitric oxide synthase immunohistochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry in pig small intestinal wholemounts revealed a complete colocalisation of the two nitrergic markers in submucous neurons. The external submucous plexus (ESP) contained nitrergic neurons throughout. In the internal submucous plexus (ISP) we found a moderate number of nitrergic neurons in the duodenum, while they were rare in the jejunum and nearly absent in the ileum. Combined NADPHd histochemistry and silver impregnation showed morphological ESP type III and VI neurons to be NADPHd positive whereas ESP type II, IV and V neurons were NADPHd negative. Axons of ESP type III, IV and VI neurons were often observed to enter interconnecting strands directed abluminally. ESP type II neurons projected mainly to the ISP. In special silver-impregnated wholemounts containing both external muscle layers and the abluminal part of the submucous layer, i.e. the myenteric plexus and the ESP, the great majority of impregnated axons within the interconnecting strands were observed to run between both plexuses and did not enter the circular muscle layer. We conclude that ESP type III and VI neurons are nitrergic while ESP type II, IV and V neurons are non-nitrergic. Furthermore, we assume that ESP type III, IV and VI neurons may represent a submucosal input to the myenteric plexus. Accepted: 26 August 1997     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Vascularization of plexus myentericus (Auerbach) in the large intestine of the cat

1. Coincidental preparation of the intramuscular vascular bed and the plexus myentericus (Auerbach) of the cat's large intestine by India-ink method and silverimpregnation allowed to demonstrate independent vascularisation of ganglia and nerve-branches of the plexus Auerbach. 2. Each ganglion is surrounded by a capillary network widely independently existing of the intramuscular capillary bed. The preferred innervated terminal arterioles and especially the sphincteric capillaries opening into the periganglionic capillary network and the numerous arterio-venous short-circuits in its marginal area suggest to conclude a differentiated regulation of blood supply.