Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

The kinetic equation for the chloride transport cycle of band 3. A 35Cl and 37Cl NMR study

The nature of a transmembrane transport process depends largely on the identity of the reaction that is rate-limiting in the transport cycle. The one-for-one exchange of two chloride ions across the red cell membrane by band 3 can be decomposed into two component reactions: 1) the binding and dissociation of chloride at the transport site, and 2) the translocation of bound chloride across the membrane. The present work utilizes 35 Cl NMR and 37 Cl NMR to set lower limits on the rates of chloride binding and dissociation at the saturated inward- and outward-facing band 3 transport sites (greater than or equal to 10(5) events site-1 s-1 in all cases). At both 0-3 and 37 degrees C, the NMR data specify that chloride binding and dissociation at the saturated transport sites are not rate-limiting, indicating that translocation of bound chloride across the membrane is the slowest step in the overall transport cycle. Using these results, it is now possible to describe many features of the kinetic equation for the ping-pong transport cycle of band 3. This transport cycle can be decomposed into two half-reactions associated with the transport of two chloride ions in opposite directions across the membrane, where each half-reaction is composed of sequential binding, translocation, and dissociation events. One half-reaction contains the rate-limiting translocation event that controls the turnover of the transport cycle; in this half-reaction, translocation must be slower than binding and dissociation. The other half-reaction contains the non-rate-limiting translocation event that in principle could be faster than binding or dissociation. However, when the following sufficient (but not necessary) condition is satisfied, both translocation events are slower than binding and dissociation: if the non-rate-limiting translocation rate is within a factor of 10(2) (0-3 degrees C) or 2 (37 degrees C) of the overall turnover rate, then translocation is rate-limiting in each saturated half-reaction. Thus, even though chloride appears to migrate through a channel that leads from the transport site to solution, the results support a picture in which the binding, dissociation, and channel migration events are rapid compared to the translocation of bound chloride across the membrane. In this case, chloride binding to the transport site can be described by a simple dissociation constant (KD = kappa OFF/kappa ON) rather than by a Michaelis-Menten constant (KM = (kappa OFF + kappa TRANSLOCATION)/KAPPA ON).     

The C2 domain calcium-binding motif: structural and functional diversity.

The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding.

The Escherichia coli D-galactose and D-glucose receptor, an aqueous periplasmic receptor that triggers sugar sensing and transport, possesses a single Ca2+ binding site similar in structure and specificity to the EF-hand class of sites found in eukaryotic Ca2+ signaling proteins including calmodulin and its homologues. A universal feature of these sites is the use of a pentagonal bipyramidal array of seven oxygens to coordinate bound Ca2+. Here we investigate the mechanisms used by this coordinating array to control ion specificity. To vary the cavity size and charge of the array, we have replaced axial glutamine 142 in the prokaryotic site with asparagine, glutamate, and aspartate. The ion selectivities of the resulting engineered sites have been quantitated by measuring dissociation constants for a series of spherical metal ions, differing in increments of radius and charge, from groups Ia, IIa, and IIIa and the lanthanides. Dramatic specificity changes are observed: sites containing an engineered smaller side chain (Asn or Asp) bind the largest cations up to 50-fold more tightly than the native site; and sites containing an engineered negative side chain (Glu or Asp) exhibit preferences for trivalent over divalent cations up to 1900-fold higher than the native site. The results indicate that the cavity size and negative charge of the coordination array play key roles in selective Ca2+ binding and that the array can be engineered to preferentially bind other cations.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Digital transformation—life cycle assessment of digital services,multifunctional devices and cloud computing

The International Journal of Life Cycle Assessment -     

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

The glycyl-radical enzyme 2-ketobutyrate formate-lyase,TdcE, interacts specifically with the formate-translocating FNT-channel protein FocA

Formate is a major product of mixed-acid fermentation in Escherichia coli. Because formate can act as an uncoupler at high concentration it must be excreted from the cell. The FNT (formate-nitrite transporter) membrane channel FocA ensures formate is translocated across the cytoplasmic membrane. Two glycyl-radical enzymes (GREs), pyruvate formate-lyase (PflB) and 2-ketobutyrate formate-lyase (TdcE), generate formate as a product of catalysis during anaerobic growth of Escherichia coli. We demonstrate in this study that TdcE, like PflB, interacts specifically with FocA. His-tagged variants of two other predicted GREs encoded in the genome of E. coli were over-produced and purified and were shown not to interact with FocA, indicating that interaction with FocA is not a general property of GREs per se. Together, these data show that only the GREs TdcE and PflB interact with the FNT channel protein and suggest that, like PflB, TdcE can control formate translocation by FocA.