Incorporation of photosensitive fatty acids into phospholipids of Escherichia coli and irradiation-dependent cross-linking of phospholipids to membrane proteins

In an approach to the study of phospholipid-protein interactions in biological membranes, the photoactivable fatty acids, omega-(m-azidophenoxy)-undecanoic acid (I) and omega-(m-diazirinophenoxy)-hexadecanoic acid (II), were incorporated biosynthetically into the phospholipids of the Escherichia coli fatty acid auxotroph, strain K1060-B5. The extent of incorporation of the two fatty acids was 43% and 21%, respectively, of the total fatty acid content of the phospholipids. Membrane vesicles prepared from cells grown on the fatty acid supplements and [32P]H3PO4 were irradiated at suitable wavelengths to generate the reactive nitrene or carbene intermediates. Subsequent analysis of solubilized membrane proteins by two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis indicated cross-linking between radioactive phospholipids and an number of proteins. A corresponding experiment with cells grown on oleic acid showed only trace amounts of covalently cross-linked phospholipid-protein adducts. While the extent of cross-linking in vesicles from cells grown on I was only 3 times the background level observed for oleic acid-grown cells, cells grown on II showed 30 times this amount. The present results, together with the previously observed nonreactivity of the nitrene generated from I to undergo C-H insertion, show that the use of carbene precursors such as II is promising for chemical analysis of specific phospholipid-protein interactions in bacterial membranes under biologically meaningful conditions.     

Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

The TrkB receptor tyrosine kinase (RTK) is a high affinity receptor for the neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Following exposure to BDNF or NT-4/5, TrkB is autophosphorylated on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Based on crystallographic analyses for others RTKs, TrkB tyrosines Y670, Y674, and Y675 are expected to lie within a putative kinase activation loop. Phosphorylation of these activation loop tyrosines is postulated to be a conserved event required for complete RTK activation. Here, we have assessed the importance these activation loop tyrosines play in regulating TrkB autophosphorylation, cytoplasmic signal transduction, and cell proliferation. We show that while tyrosine 670 is dispensable for BDNF-inducible TrkB autophosphorylation and the activation of certain signal transduction events, it is required for complete TrkB-mediated cellular proliferation. Combinatorial mutagenesis of tyrosines 674 and 675 only moderately affects TrkB autophosphorylation, but significantly impairs the BDNF-inducible stimulation of cytoplasmic signaling events and cellular proliferation. The combined mutation of all three activation loop tyrosines results in an inactive receptor, which is unable to autophosphorylate, stimulate signaling events, or induce mitogenesis. The data highlight the varying degrees of importance of the three activation loop tyrosines in TrkB mediated biological responses.     

Agreement between clinical examination and parental morbidity histories for children in Nepal

Traumatic diaphragmatic hernia is rare, but is of utmost importance due to its high morbidity and mortality. It is markedly important in patients with blunt abdominal trauma, and diagnosis is difficult because of the numerous associated injuries. A patient with few symptoms of chronic traumatic diaphragmatic hernia is described, who underwent surgery due to a gastric volvulus. Laparoscopic surgery permits repair of these injuries through an abdominal approach, avoiding a thoracic incision or selective intubation.     

Percutaneous biopsy of small hepatic lesions using an 18 gauge automated needle

This study evaluates the efficacy of ultrasound guided percutaneous biopsy using an 18 gauge automated side-cutting needle in the diagnosis of small (< or = 3 cm) focal hepatic lesions. 137 consecutive percutaneous biopsies of 131 different small (< or = 3 cm) focal hepatic lesions were included. 11 biopsies were performed on lesions of < or = 1 cm in diameter, 56 were on lesions 1.1-2 cm in size and 70 on lesions 2.1-3 cm in size (average 2.3 +/- 0.7 cm; median 2.3 cm; range 0.7-3 cm). The biopsy specimen was sufficient for diagnosis in 135 cases (98.5%). The sensitivity for diagnosing malignancy was 96.4%; specificity was 100%, positive and negative predictive values were 100% and 94.6%, respectively; accuracy was 97.8%. There was no statistically significant difference in the diagnostic efficacy for lesions of different pathology and size. No significant post-biopsy complication occurred. It is concluded that the 18 gauge Temno needle is safe and effective in diagnosing small hepatic lesions.     

Involvement of capsaicin sensitive primary afferents in thymulin-induced hyperalgesia

Intraplantar (5 ng) or intraperitoneal (50 ng) injections of thymulin, produced both thermal and mechanical hyperalgesia in rats. In this report, we show that ablation of capsaicin sensitive primary afferents (CSPA) can alter or abolish thymulin-induced hyperalgesia. Different groups of rats were subjected to either treatment with capsaicin or to surgical subdiaphragmatic vagotomy (SDV). Both capsaicin and SDV reduced significantly thymulin-induced hyperalgesia. On the other hand, these treatments elicited differential effects on the modulation by thymulin of the levels of nerve growth factor and interleukin 1beta. We conclude that the hyperalgesic effects of i.p. thymulin are mainly mediated through the CSPA fibers.     

Arterial vascular remodeling: the endothelial cell''s central role

As a measurement of the level of anxiety in psychiatric outpatients with anxiety, we determined the saliva level of free 3-methoxy-4-hydroxyphenyleglycol (MHPG) using gas chromatography- mass spectrometry and scored the levels of anxiety with the Hamilton Anxiety Scale (HAS) in patients, before and after drug treatment with alprazolam for 1 week. The saliva level of free-MHPG at first visit to hospital was significantly higher than that of control individuals and disease control individuals and was reduced by alprazolam treatment for 1 week. There was no correlation between MHPG level and the HAS score at the first hospital visit. The MHPG levels after treatment correlated with the HAS scores. The reduction of the anxiety level as scored by the HAS correlated with the reduction of MHPG level. These results indicate that the free saliva MHPG level may be a useful indicator for assessing not only the level of anxiety, but also the response to drug treatment for anxiety in these patients.     

Laminin and alpha-dystroglycan mediate acetylcholine receptor aggregation via a MuSK-independent pathway

Specific isoforms of laminin (LN) are concentrated at neuromuscular junctions (NMJs) where they may participate in synaptic organization or function. In myotubes from C2 cells, LN is concentrated within the majority of spontaneous acetylcholine receptor (AChR) aggregates. Neural agrin substantially increases this colocalization, suggesting that agrin can recruit LN into AChR aggregates. Addition of LN to C2 myotubes induces a more than twofold increase in the number of AChR aggregates. These aggregates have a larger size and are more dense than are those induced by agrin, suggesting that LN is involved in the growth and/or stabilization of AChR aggregates. Consistent with this hypothesis, an antiserum to LN reduces the size of individual AChR aggregates but increases their number. In C2 myotubes, extracellular matrix receptors containing the integrin beta1 subunit are poorly colocalized with AChR aggregates, suggesting that integrins may not be involved in LN-induced aggregation. In contrast, almost all AChR aggregates are associated with dystroglycan immunoreactivity, and monoclonal antibody (mAb) IIH6 against alpha-dystroglycan (alpha-DG), a LN and agrin receptor, causes a concentration-dependent inhibition of LN-induced aggregation. Moreover, S27 cells, which lack a functional alpha-DG, and two C2-derived cell lines expressing antisense DG mRNA fail to aggregate AChRs in response to LN. Finally, LN-induced AChR aggregation does not involve the phosphorylation of the muscle-specific tyrosine kinase receptor (MuSK) or the AChR beta subunit. We hypothesize that the interaction of LN with alpha-DG contributes to the growth and/or stabilization of AChR microaggregates into macroaggregates at the developing NMJ via a MuSK-independent mechanism.     

Promiscuous coassembly of serotonin 5-HT3 and nicotinic alpha4 receptor subunits into Ca(2+)-permeable ion channels

Serotonin (5-hydroxytryptamine) type 3 receptors (5-HT3R) and nicotinic acetylcholine receptors are structurally and functionally related proteins, yet distinct members of the family of ligand-gated ion channels. For most members of this family a diversity of heteromeric receptors is known at present. In contrast, known 5-HT3R subunits are all homologs of the same 5-HT3R-A subunit and form homopentameric receptors. Here we show, by heterologous expression followed by immunoprecipitation, that 5-HT3R and nicotinic acetylcholine receptor alpha4 subunits coassemble into a novel type of heteromeric ligand-gated ion channel, which is activated by 5-HT. The Ca2+ permeability of this heteromeric ion channel is enhanced as compared with that of the homomeric 5-HT3R channel. Heteromeric 5-HT3/alpha4 and homomeric 5-HT3Rs have similar pharmacological profiles, but distinct sensitivities to block by the antagonist d-tubocurarine. Coassembly of subunits beyond the boundaries of ligand-gated ion channel families may constitute an important mechanism contributing to the diverse properties and functions of native neurotransmitter receptors.