Model for the electrolytic environment and electrostatic properties of biomembranes

Physical and chemical interactions of ions with biomembranes are described by a model originating from the Stern theory. Equations of the model have analytical solutions only for very simple, often unrealistic situations. The numerical resolution adopted permits a much wider application of the model: Potentials and concentrations can be calculated anywhere from the surface and in any electrolytic environment. The model is applied to biomembranes. Simulations are presented in three-dimensional figures which allow one to use the model as a practical research tool. In particular, the simulations reveal that, in practice, it is possible to induce an increase of the surface charge density simultaneously with a decrease of the surface potential, and, theoretically, that the potential at the exclusion distance (which estimates the diffuse layer thickness) exhibits a remarkably constant value as the composition of the free solution is varied.     

A case study employing operant conditioning to reduce stress of capture for red-bellied tamarins (Saguinus labiatus)

Traditional techniques used to capture New World monkeys, such as net capture, can induce high levels of acute stress detrimental to welfare. Alternatively, training nonhuman animals via operant conditioning to voluntarily participate in husbandry and/or veterinary practices is accepted as a humane process that can reduce stress and improve welfare. This study details the use of operant conditioning using positive reinforcement training (PRT) and target training to train a family of 5 captive red-bellied tamarins (Saguinus labiatus) in a wildlife park to voluntarily enter a transportation box and remain calm for 1 min after 54 training sessions. Observations of 2 unrelated net-capture processes provided measures of locomotion and vocalizations as indicators of stress behavior that were compared with those of the trained tamarins. Net-captured monkeys exhibited rapid erratic locomotion and emitted long, high-frequency vocalizations during capture whereas the trained tamarins exhibited minimal locomotion and emitted only 4 brief vocalizations (root mean square 35 dB) during capture. This indicates that the use of PRT considerably reduced potential for stress and improved welfare during the capture and containment of the tamarins.     

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.     

Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G,determines the specificity of RSV infection

Human respiratory syncytial virus (HRSV) and bovine RSV (BRSV) infect human beings and cattle in a species-specific manner. We have here analyzed the contribution of RSV envelope proteins to species-specific entry into cells. In contrast to permanent cell lines, primary cells of human or bovine origin, including differentiated respiratory epithelia, peripheral blood lymphocytes, and macrophages, showed a pronounced species-specific permissiveness for HRSV and BRSV infection, respectively. Recombinant BRSV deletion mutants lacking either the small hydrophobic (SH) protein gene or both SH and the attachment glycoprotein (G) gene retained their specificity for bovine cells, whereas corresponding mutants carrying the HRSV F gene specifically infected human cells. To further narrow the responsible region of F, two reciprocal chimeric F constructs were assembled from BRSV and HRSV F1 and F2 subunits. The specificity of recombinant RSV carrying only the chimeric F proteins strictly correlated with the origin of the membrane-distal F2 domain. A contribution of G to the specificity of entry could be excluded after reintroduction of BRSV or HRSV G. Virus with F1 and G from BRSV and with only F2 from HRSV specifically infected human cells, whereas virus expressing F1 and G from HRSV and F2 from BRSV specifically infected bovine cells. The introduction of G enhanced the infectiousness of both chimeric viruses to equal degrees. Thus, the role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans. The identification of the F2 subunit as the determinant of RSV host cell specificity facilitates identification of virus receptors and should allow for development of reagents specifically interfering with RSV entry.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A

C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.     

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1

A protein phosphatase assay, selective for protein phosphatase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [gamma-32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10-60-times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.     

Adsorption and exchange of Ca,Mg and K-ions on the root cell walls of clover and rye-grass

Summary The ion exchange properties of clover and rye-grass root cell walls were studied quantitatively. Three sets of experiments were performed: adsorption of Ca, Mg and K ions versus pH, adsorption versus cation concentration and exchange isotherms Ca–Mg and Mg–K. A model has been developed. It allows the satisfactory prediction of results (except for pH curves) with the adjustment of a minimum of parameters. The total charge (RT), on its own, accounts for the difference between the ion exchange properties of the clover and rye grass cell walls. The selectivities observed on root material are very different from those observed on soil exchange complexes. The decreasing affinities of cell walls for cations follow the sequence: Ca>MgK for cell walls. These differences of selectivity are much larger than those usually observed for soil exchange complexes.