Morphological observations on motor end-plates in rabbits with experimental myasthenia

The morphology of motor end-plates in rabbits immunized with Torpedo nicotinic acetylcholine receptor (nAChR) has been studied by light and electron microscopy. Rabbits were studied either after one period of paralysis, some in parallel with electrophysiological recordings of MEPPs and EPPs and of Naja naja alpha-neurotoxin binding properties or after recovery followed by a second paralysis. Changes in the sub-neural apparatus were noted after cholinesterase staining only in the latter group. Ultrastructurally, however, most end-plates in both groups contained a wide range of abnormalities. Many were similar in appearance to those observed in human myasthenia gravis (MG). This further supports the theory that immunized rabbits can be used as a model for myasthenia gravis. In the rabbits with 1 period of paralysis an acute stage of influence on the neuromuscular junction seemed to be present while simplified motor end-plates typical for human MG were mostly found in rabbits with 2 periods of paralysis. Short post-synaptic folds in conjugation with thickeneed membrane-bound vesicles at their tops, inside the basement membrane, were frequently observed. These were interpreted as if the crests of the folds containing nAChR had degenerated and had been budded off. If so, a large number of receptor sites had been lost which would be one possible explanation for the lowered capacity of the muscles to bind Naja naja alpha-neurotoxin. Membrane thickenings with projections and striations were interpreted as reflecting ACh receptors and were observed in the post-junctional membrane without proximity to the nerve terminal. The degeneration of the top of the post-synaptic folds and the occurrence of receptors at other locations within the motor end-plate will result in a widened distance between the nerve terminal and the receptors, which can explain previous interpretations of a presynaptic defect in MG.     

Assessment of the arachidonic acid content in foods commonly consumed in the American diet

Arachidonic acid (AA) is an extremely important fatty acid involved in cell regulation. When provided in the diet, it is cogently incorporated in membrane phospholipids and enhances eicosanoid biosynthesis in vivo and in vitro; however, controversy exists as to the levels of AA in food and in the diet. This study determined the amount of AA in cooked and raw portions of beef (rib eye), chicken (breast and thigh), eggs, pork (loin), turkey (breast), and tuna; it compared these results to values published in Agriculture Handbook No. 8 (HB-8). The cooked portions were prepared as described in HB-8. With the exception of chicken thigh and tuna, the levels of AA (w/w) in the selected foods analyzed were significantly higher, in general, than those values published in HB-8. The greatest differences were observed in beef (raw and cooked), turkey breast (raw and cooked), and pork (cooked) where AA levels were twice that of the values in HB-8. In contrast, the AA and n-3 fatty acid contents in tuna were almost half the HB-8 values. The present data indicate that HB-8 tends to underreport the amounts of AA in a number of foods commonly consumed in the American diet, and new initiatives should be considered to validate and update the current database for fatty acid composition of foods.     

Intracochlear factors contributing to psychophysical percepts following cochlear implantation

Glycosyl-phosphatidylinositol (GPI) lipids have a structural role as protein anchors to the cell surface. In addition, they are implicated in hormone, growth factor and cytokine signal transduction. Their phosphodiesteric hydrolysis mediated by an activated phospholipase results in the generation of water soluble oligosaccharide species termed the inositol phosphoglycan (IPG). This product has been demonstrated to possess biological properties when added exogenously to cells mimicking the biological effects of a variety of extracellular ligands. This may be accomplished since IPG is generic for a family of closely related species which are released in a tissue-specific manner and additionally have cell-specific targets. Micro-organic synthesis has recently been able to shed new light on this topic by the introduction of defined oligosaccharide analogues of IPG for the assessment of their biological activity. These have complemented the findings observed with purified IPG from biological sources thus strengthening the belief that the GPI/IPG signalling system represents a truly novel aspect of transmembrane signalling.     

Racial differences in the distribution of a low density lipoprotein receptor-related protein (LRP) polymorphism and its association with serum lipoprotein, lipid and apolipoprotein levels

We recently demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) is an autocrine growth factor for human osteoblastic (hOB) cells. Since GM-CSF is a member of the heparin-binding factor family, we examined the interactions between GM-CSF and glycosaminoglycans (GAGs) present in the osteoblast microenvironment. Using a bioassay in which the mitogenic activity of recombinant human (rh) GM-CSF was measured after incubation in the presence of an hOB cell layer or extracellular matrix (ECM) produced by these cells, we showed that rhGM-CSF binds to GAG components present in the ECM and that the bound rhGM-CSF retains its ability to stimulate hOB cell proliferation. Heparan sulfate compounds on the hOB cell surface were also found to sequester GM-CSF. Moreover, treatment with sodium chlorate, an inhibitor of GAG sulfation, suppressed the mitogenic activity of rhGM-CSF on hOB cells. This inhibitory effect was rescued by a low dose of heparin. Heparin was also found to promote the effect of rhGM-CSF on hOB cell proliferation, allowing nonmitogenic high doses of rhGM-CSF to stimulate hOB cell growth. Western blot analysis showed that undersulfation of cellular GAGs by chlorate inhibited the increased tyrosine phosphorylation of proteins involved in GM-CSF signaling in cloned immortalized hOB cells. The data demonstrate that GM-CSF binds to proteoglycans on the hOB cell surface and in ECM produced by these cells and that the bound GM-CSF is biologically active. Furthermore, this study shows that cellular proteoglycans play an essential role in GM-CSF signaling and biological activity in hOBs.     

Transforming growth factor beta1 induces nuclear export of inhibitory Smad7

Transforming growth factor beta (TGF-beta) signals from membrane to nucleus through serine/threonine kinase receptors and their downstream effector molecules, termed Smad proteins. Recently, Smad6 and Smad7 were identified, which antagonize TGF-beta family signaling by preventing the activation of signal-transducing Smad complexes. Here we report that Smad7, but not Smad6, inhibits TGF-beta1-induced growth inhibition and the expression of immediate early response genes, including Smad7. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-beta receptor activation. The latter is in accordance with the physical association of Smad7 with the ligand-activated TGF-beta receptor complex in the cell membrane. Whereas the ectopically expressed C-terminal domain of Smad7 was also exported from the nucleus to the cytoplasm upon TGF-beta challenge, a Smad7 mutant with a small deletion at the C terminus or only the N-terminal domain of Smad7 was localized mainly in the cytoplasm in the absence or presence of ligand. This suggests that an intact Mad homology 2 domain is important for nuclear localization of Smad7. The nuclear localization of Smad7 suggests a functional role distinct from its antagonistic effect in receptor-mediated Smad activation.     

Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA

A lysate of human herpesvirus type 6 (HHV6) infected HSB2 cells was used as antigen for an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibody to HHV6. 78 clinical samples were tested for the presence of HHV6-specific IgM. Nine specimens, all from children under 4.5 years of age, were found to be reactive indicating probable acute infection with HHV6. Sera from 12 healthy adult blood donors and from 88 of 90 adults over the age of 35 with unspecified health conditions tested negative for HHV6 IgM, indicating a minimum specificity estimate of nearly 98% in these patients. Cross-reactivity of antibody to other herpes viruses with the HHV6 ELISA antigen was not detected. Six hundred and ninety-six serum samples from individuals of different age groups were examined for IgG antibody status. In 94% of these samples, IgG antibody was detected. Our data suggests that most Canadians possess antibody to HHV6 by 1 yr of age and that on average, antibody levels remain high through early adulthood but begin to decline with advancing age. The ELISA described is a reliable test for the measurement of IgG and IgM antibodies for both clinical diagnosis and epidemiological studies.     

A correlative study on the mechanism of adaptive cytoprotection against ethanol-induced gastric lesion formation in rats

The protective effect of mild irritants against the subsequent gastric injury induced by necrotizing agents has been termed 'adaptive cytoprotection'. In this study, the possible pathway and mechanisms of adaptive cytoprotection induced by 20% ethanol were investigated. An ex-vivo gastric chamber preparation was used. The gastric mucosa was exposed to 20% ethanol before subsequent administration of 100% ethanol 15 min later. Subdiaphragmatic vagotomy or drug pretreatment was carried out in order to elucidate the mechanisms of adaptive cytoprotection by 20% ethanol. The results showed that 20% ethanol pre-exposure significantly protected the gastric mucosa against damage caused by 100% ethanol. This protective action was completely abolished by atropine or lidocaine pretreatment, whereas vagotomy and hexamethonium failed to have a significant influence. The cytoprotective effect, however, was independent of the gastric secretory volume, titratable acid content, luminal soluble mucus level and gastric mucosal blood flow. Exposure of only half the gastric mucosa to the mild irritant resulted in the protection of both sides of the mucosa. All these findings indicate that the adaptive cytoprotection of 20% ethanol involves the participation of chemoreceptors and muscarinic receptors in the gastric mucosa. An internal enteric reflex arc, with transmission of signals within the gastric mucosa, may also contribute to the cytoprotective process of the mild irritant.     

Thermodynamic and fluorescence studies of the underlying factors in benzyl alcohol-induced lipid interdigitated phase

To further investigate factors contributing to the action of alcohol in the solute-induced lipid interdigitation phase, thermodynamic and fluorescence polarization measurements were carried out to study the interaction of benzyl alcohol with dipalmitoyl phosphatidylcholine bilayer vesicles. The obtained results were compared with those previously reported for ethanol and cyclohexanol (L. G. Roth and C-H. Chen, Arch. Biochem. Biophys. 296, 207, 1992). Similar to ethanol, benzyl alcohol was found to exhibit a biphasic effect on the enthalpy (delta Hm) and the temperature (tm) of the lipid-phase transition and the steady-state fluorescence polarization (P) monitored by 1,6-diphenyl-1,3,5-hexatriene. At a total concentration of benzyl alcohol < 30 mg/ml (the alcohol concentration in lipid phase < 21 mg/ml), benzyl alcohol was found to exhibit large increases in delta Hm and P, which were correlated with the formation of a lipid interdigitated phase, as evidenced by reported X-ray diffraction data. Combining the results with benzyl alcohol and ethanol suggested that simultaneously large changes in delta Hm and P can be used as an indication of the occurrence of a solute-induced lipid interdigitated phase. The overall interacting force in the formation of this lipid phase, as derived from the interactions of the hydroxyl portion of an alcohol with the lipid phosphate head group and the hydrophobic portion of an alcohol with the lipid hydrocarbon chains, may or may not be dominated by hydrophobic interaction. Although lipid/water partition coefficients and the contribution of hydrophobic interaction to the overall interacting force were comparable between benzyl alcohol and cyclohexanol, benzyl alcohol induced lipid interdigitated phase, but not for cyclohexanol. This was due to the ability of benzyl alcohol to be more effective than cyclohexanol in simultaneously interacting with the phosphate head group and the hydrocarbon chains of lipid.