Kinetic Process of β-Amyloid Formation via Membrane Binding

Recently we have studied thermodynamics of membrane-mediated β-amyloid formation in equilibrium experiments using penetratin-lipid mixtures. The results showed that penetratin bound to the membrane interface in the α-helical conformation when the peptide/lipid (P/L) ratios were below a lipid-dependent critical value P/L^∗. When P/L reached P/L^∗, small β-aggregates emerged, which served as the nuclei for large β-aggregates. Here we studied the corresponding kinetic process to understand the potential barriers for the membrane-mediated β-amyloid formation. We performed kinetic experiments using giant unilamellar vesicles made of 7:3 DOPC/DOPG. The observed time behavior of individual giant unilamellar vesicles, although complex, exhibited the physical effects seen in equilibrium experiments. Most interestingly, a potential barrier appeared to block penetratin from translocating across the bilayer. As a result, the kinetic value for the critical threshold P/L^∗ is roughly one-half of the value measured in equilibrium where peptides bind symmetrically on both sides of lipid bilayers. We also investigated the similarity and differences between the charged and neutral lipids in their interactions with penetratin. We reached an important conclusion that the bound states of peptides in lipid bilayers are largely independent of the charge on the lipid headgroups.     

Enhancement of cell growth by intracellularly expressed herpes simplex virus thymidine kinase

A recombinant cell line (NIH3T3:pLtkSN) was made by infecting parental cells (NIH3T3) with a recombinant retrovirus (pLtkSN) encoding herpes simplex virus thymidine kinase (HSVtk) gene. The cells expressing HSVtk (NIH3T3:pLtkSN) grew 2.3 times more than the parental cells (NIH3T3) in Dulbecco's Modified Eagles Media containing 10% (v/v) horse serum. The NIH3T3:pLtkSN cells also showed a significant enhancement in the maximal cell concentration and the specific growth rate even at 2.5% serum concentration. The specific O2 uptake rate of NIH3T3 was 2.1 times greater than that of NIH3T3:pLtkSN. Under both O2-limited and O2-unlimited conditions, it appears that HSVtk plays an important role in enhancing the growth characteristics of animal cells.     

A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.     

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.     

Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.