Interaction of the beta-adrenergic receptor with Gs following delipidation. Specific lipid requirements for Gs activation and GTPase function

Preparations of beta-adrenergic receptor and Gs from turkey erythrocytes were delipidated by previously developed procedures. Three synthetic phospholipids, dioleoylglycerophosphoethanolamine, dioleoylglycerophosphocholine and dioleoylglycerophosphoserine plus an unphosphorylated lipid, were all required to restore receptor-mediated activation of Gs by GTP[gamma S]. The same lipids were necessary for the reconstitution of the isoproterenol-enhanced GTPase. The requirement for the unphosphorylated lipid could be fulfilled by 1-mono-oleoyl glycerol, alpha-tocopherol or oleic acid. Cholesterol hemisuccinate further enhanced the receptor-mediated activity of the relipidated system when present in addition to the lipids specified above. Cholesterol hemisuccinate had no effect on the basal rate of Gs activation and depressed the basal GTPase. It is therefore suggested that cholesterol hemisuccinate affects the receptor or the coupling of the receptor to Gs. In the system relipidated with the three dioleoyl phospholipids, plus alpha-tocopherol and cholesterol hemisuccinate, the initial rate of Gs activation per mole receptor appeared to be considerably higher than in the native turkey erythrocyte membrane.     

Mise en évidence de polysaccharides par le noir Soudan B après estérification

Résumé Les effets de l'acétylation réversible et de l'estérification sulfurique sur la coloration de polysaccharides par le noir Soudan B sont étudiés après inclusion à la paraffine; les faits suivants sont à noter. 1° Une affinité nette pour le noir Soudan B en solution alcoolique existe, dans le cas des polysaccharides très acides, sans aucun prétraitement des coupes. 2° Tous les polysaccharides présentent, après acétylation ou estérification sulfurique, une forte affinité pour le noir Soudan B, le bleu alcian et le bleu de toluidine; la teinte est souvent métachromatique avec ce dernier colorant. 3° L'affinité des polysaccharides pour les trois colorants disparaît après saponification des coupes acétylées. 4° Les teintes les plus intenses sont obtenues avec les solutions vieillies ou acétifiées de noir Soudan B. — Ces faits plaident en faveur de l'hypothèse suivant laquelle le noir Soudan B possède certaines propriétés des colorants basiques.

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Staining of polysaccharides with Sudan black B after esterification

Summary This study centers on the effects of acetylation, deacetylation, and sulphation on the staining of polysaccharides with Sudan black B in paraffin sections; it brings up the following facts: A definite staining of strongly acidic polysaccharides with alcoholic Sudan black B is obtained without any pretreatment of the sections. — After acetylation or sulphation, all the investigated polysaccharides exhibit a strong affinity to Sudan black B, alcian blue and toluidine blue; a distinct metachromasia is obtained with the third of these stains. — Any affinity of polysaccharides to the three dyestuffs is suppressed by deacetylation of the acetylated sections. — The most intense staining with Sudan black B is given by old or acetified solutions. — These data are consistent with the hypothesis according to which Sudan black B is provided with some characteristics of a basic stain.

     

A weakened interface in the P182L variant of HSP27 associated with severe Charcot‐Marie‐Tooth neuropathy causes aberrant binding to interacting proteins

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions.     

Isoflurane blocks glutamatergic excitatory transmission pre- and postsynaptically in crayfish muscle

Crayfish neuromuscular junctions are good models for the α-amino-hydroxy-5-methyl-4-isoxazol-propionic acid-type of vertebrate brain excitatory synapses. The action of a typical volatile anaesthetic, isoflurane, was studied on the excitatory postsynaptic currents recorded with a perfused macropatch electrode. Isoflurane reduced quantal exitatory postsynaptic currents in amplitude, in their rise time and in the decay time constant. Small such effects were elicited by <1 mmol · l⁻¹ isoflurane, while the maximal isoflurane concentration of 7 mmol · l⁻¹ reduced the amplitude to about a quarter and shortened the decay time constant even more, while the rise time was diminished by about a quarter. This combination of effects is typical for an open channel block for which an approximate binding rate constant of isoflurane of 6 · 10⁵ mol⁻¹l · s⁻¹ and an unbinding rate of 10–100 s⁻¹ is derived. In addition to this postsynaptic effect, isoflurane inhibited the release of transmitter quanta from the terminal, for instance with 2.5 mmol · l⁻¹ isoflurane by a factor of 7.3 ± 6.3 (SD). In the glutamatergic nerve terminals release is modulated by low glutamate concentrations via a metabotropic autoreceptor which is blocked by the combination of 6-cyano-7-nitro-quinoxaline-2,3-dione and dl-2-amino-5-phosphor-valeric acid. This blocker combination also can prevent the inhibition of release by isoflurane, and it may be suggested that isoflurane elicits inhibition of release through the metabotropic presynaptic glutamate receptors. Accepted: 29 March 1998     

Am Jadebusen

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Aus Brandenburg

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A new stable epithelial cell line (RK-L) from normal rat kidney

Summary A stable epithelial cell line has been established from the kidneys of a normal Sprague-Dawley rat. This line, termed RK-L, has a high proliferative capacity (minimal doubling time 12.3 h) and can be grown in medium containing 1% fetal bovine serum. Thus far, the line has been carried through more than 60 serial passages. The RK-L cells were found to display similarities with kidney tubule cells. Using light microscopy, confluent cultures were seen as pavement-like monolayers forming domes, which are thought to result from transepithelial fluid transport. Electron microscopy revealed polarized cells that had microvilli on the apical surface, junction complexes in the apical part of the lateral cell membrane, and a basal lamina-like layer. Pinocytotic activity was indicated by infoldings of the apical plasma membrane and the formation of vesicles. The RK-L line should prove useful for investigations of kidney tubule transport mechanisms.     

Botanische Erinnerungen eines Nord-Deutschen aus der Gegend von Triest

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