A continuous-binding cross-linker model for passive airway smooth muscle

Although the active properties of airway smooth muscle (ASM) have garnered much modeling attention, the passive mechanical properties are not as well studied. In particular, there are important dynamic effects observed in passive ASM, particularly strain-induced fluidization, which have been observed both experimentally and in models; however, to date these models have left an incomplete picture of the biophysical, mechanistic basis for these behaviors. The well-known Huxley cross-bridge model has for many years successfully described many of the active behaviors of smooth muscle using sliding filament theory; here, we propose to extend this theory to passive biological soft tissue, particularly ASM, using as a basis the attachment and detachment of cross-linker proteins at a continuum of cross-linker binding sites. The resulting mathematical model exhibits strain-induced fluidization, as well as several types of force recovery, at the same time suggesting a new mechanistic basis for the behavior. The model is validated by comparison to new data from experimental preparations of rat tracheal airway smooth muscle. Furthermore, experiments in noncontractile tissue show qualitatively similar behavior, suggesting support for the protein-filament theory as a biomechanical basis for the behavior.     

GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain

We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down-stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration- and time-dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region-specific distribution. Adding GM1 to brain slices activated extracellular-regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time-and concentration-dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1-induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein-coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.     

IP(3)-induced tension and IP(3)-receptor expression in rat soleus muscle during postnatal development

The present study was designed to examine whether changes in Ca(2+) release by inositol-1,4,5-trisphosphate (IP(3)) in 8-, 15-, and 30-day-old rat skeletal muscles could be associated with the expression of IP(3) receptors. Experiments were conducted in slow-twitch muscle in which both IP(3)-induced Ca(2+) release and IP(3)-receptor (IP(3)R) expression have been shown to be larger than in fast-twitch muscle. In saponin-skinned fibers, IP(3) induced transient contractile responses in which the amplitude was dependent on the Ca(2+)-loading period with the maximal IP(3) contracture being at 20 min of loading. The IP(3) tension decreased during postnatal development, was partially inhibited by ryanodine (100 microM), and was blocked by heparin (20-400 microg/ml). Amplification of the DNA sequence encoding for IP(3)R isoforms (using the RT-PCR technique) showed that in slow-twitch muscle, the type 2 isoform is mainly expressed, and its level decreases during postnatal development in parallel with changes in IP(3) responses in immature fibers. IP(3)-induced Ca(2+) release would then have greater participation in excitation-contraction coupling in developing fibers than in mature muscle.     

Variegate Porphyria in Western Europe: Identification of PPOX Gene Mutations in 104 Families, Extent of Allelic Heterogeneity, and Absence of Correlation between Phenotype and Type of Mutation

Variegate porphyria (VP) is a low-penetrance, autosomal dominant disorder characterized clinically by skin lesions and acute neurovisceral attacks that occur separately or together. It results from partial deficiency of protoporphyrinogen oxidase encoded by the PPOX gene. VP is relatively common in South Africa, where most patients have inherited the same mutation in the PPOX gene from a common ancestor, but few families from elsewhere have been studied. Here we describe the molecular basis and clinical features of 108 unrelated patients from France and the United Kingdom. Mutations in the PPOX gene were identified by a combination of screening (denaturing gradient gel electrophoresis, heteroduplex analysis, or denaturing high-performance liquid chromatography) and direct automated sequencing of amplified genomic DNA. A total of 60 novel and 6 previously reported mutations (25 missense, 24 frameshift, 10 splice site, and 7 nonsense) were identified in 104 (96%) of these unrelated patients, together with 3 previously unrecognized single-nucleotide polymorphisms. VP is less heterogeneous than other acute porphyrias; 5 mutations were present in 28 (26%) of the families, whereas 47 mutations were restricted to 1 family; only 2 mutations were found in both countries. The pattern of clinical presentation was identical to that reported from South Africa and was not influenced by type of mutation. Our results define the molecular genetics of VP in western Europe, demonstrate its allelic heterogeneity outside South Africa, and show that genotype is not a significant determinant of mode of presentation.     

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

Background

Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.

Methods

To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (F_unb) in the absence and presence of calponin or phosphorylated calponin.

Results

F_unb from F-actin alone (0.12 ± 0.01 pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02 pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01 pN). To further verify that this enhancement of F_unb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the F_unb obtained at a [KCl] of 25 mM (0.21 ± 0.02 pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01 pN).

Conclusions

This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.

General significance

This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.     

Enzymatic/biochemical analysis of Actinomyces with commercial test kits with an emphasis on newly described species

In clinical microbiology laboratories, the identification of Actinomyces-like bacteria can be very laborious and problematic. In the present study, we focused on reactivity patterns of 4 commercial test kits, RapID ANA II, RapID 32A, RapID CB Plus, and BBL Crystal ANR ID, that could be used for rapid preliminary identification of Actinomyces isolates belonging to newly described Actinomyces and closely related species. Out of the 54 strains tested, 25 strains (46%) were correctly identified to the genus/group level by BBL Crystal ANR ID system, 16 strains (30%) by RapID 32 A, 11 strains (20%) by RapID CB Plus, and 7 strains (13%) by RapID ANA II. The main problems with these kits were due to occasional weak enzymatic and sugar fermentation reactions. In conclusion, chromogenic substrate sensitivity and specificity need to be enhanced in order to improve the reliability of the test results of these kits, and the present database updated in order to more precisely identify newly described Actinomyces and closely related species.     

Selective defect in antigen-induced TCR internalization at the immune synapse of CD8 T cells bearing the ZAP-70(Y292F) mutation

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3zeta degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.     

Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase

Guanosine monophosphate kinases (GMPKs), which catalyze the phosphorylation of GMP and dGMP to their diphosphate form, have been characterized as monomeric enzymes in eukaryotes and prokaryotes. Here, we report that GMPK from Escherichia coli (ecGMPK) assembles in solution and in the crystal as several different oligomers. Thermodynamic analysis of ecGMPK using differential scanning calorimetry shows that the enzyme is in equilibrium between a dimer and higher order oligomers, whose relative amounts depend on protein concentration, ionic strength, and the presence of ATP. Crystallographic structures of ecGMPK in the apo, GMP and GDP-bound forms were solved at 3.2A, 2.9A and 2.4A resolution, respectively. ecGMPK forms a hexamer with D3 symmetry in all crystal forms, in which the two nucleotide-binding domains are able to undergo closure comparable to that of monomeric GMPKs. The 2-fold and 3-fold interfaces involve a 20-residue C-terminal extension and a sequence signature, respectively, that are missing from monomeric eukaryotic GMPKs, explaining why ecGMPK forms oligomers. These signatures are found in GMPKs from proteobacteria, some of which are human pathogens. GMPKs from these bacteria are thus likely to form the same quaternary structures. The shift of the thermodynamic equilibrium towards the dimer at low ecGMPK concentration together with the observation that inter-subunit interactions partially occlude the ATP-binding site in the hexameric structure suggest that the dimer may be the active species at physiological enzyme concentration.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.