Structure and expression of the herpes simplex virus type 2 glycoprotein gB gene.

The gene for glycoprotein gB2 of herpes simplex virus type 2 strain 333 was cloned, sequenced, and expressed in mammalian cells. The gB2 protein had an overall nucleotide and amino acid sequence homology of 86% with the cognate gB1 protein. However, of the 125 amino acid substitutions or deletions, only 12.5% were conservative replacements. These differences were clustered within an NH2-terminal region, a central region, and a COOH-terminal region, resulting in domains of near identity broken by small regions of marked divergence. Regions of greatest homology included a 90-amino-acid stretch starting at residue 484 and 39 amino acids spanning residues 835 to 873, which cover a rate-of-entry locus mapped to Ala-552 and a syn locus mapped to Arg-857, respectively, in gB1 by Bzik et al. (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984). Pellett et al. (P. E. Pellett, K. G. Kousoulas, L. Pereira, and B. Roizman, J. Virol. 53:243-253, 1985) mapped the mutations in three monoclonal antibody-resistant gB1 mutants between amino acids 273 and 443. These epitopes are included in a region of 98 residues identical between gB1 and gB2. The identity of this protein was verified by placing a truncated gene lacking the 303 carboxyl-terminal amino acids of gB2 into mammalian COS and CHO cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation. This protein will be purified from the stable CHO cell lines and compared with gB1 for immunogenicity and protective efficacy in animal challenge models.     

The influence of cytotoxins from Central Asian cobra venom and melittin from bee venom on the thermodynamic properties of phospholipid bilayer

The interaction of cytotoxin Vc1 and Vc5 from Central Asian cobra and melittin from the bee venom with multilayer liposomes prepared from dimiristoylphosphatidylcholine with an addition of phosphatidic acid have been studied by the method of differential scanning calorimetry. Incorporation of Vc1, Vc5 and melittin into the lipid resulted in pronounced changes in the thermodynamic properties of the lipid. Polypeptides studied induced lateral phase separation in the lipid. Interaction between molecules of the toxins and the lipid resulted in the formation of a new lipid phase characterized by a higher melting temperature and lower phase transition enthalpy.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Cooperative binding of primycin and gramicidin on erythrocyte membranes. A cation transport study

In this paper the authors present a comparative study of the actions of the antibiotics primycin and gramicidin on the erythrocyte membrane permeability. It has been found that both antibiotics have a nonlinear effect on the membrane permeability. Above a threshold antibiotic concentration, which is characteristic of the type of the antibiotic, the cation permeability of the erythrocyte membranes increases sharply. In the range of nonlinearity the transport-kinetic curves level off before achieving the equilibrium radioactive ion distribution between the extra- and intracellular spaces. A stochastic model of the cooperative and aspecific incorporation of antibiotic molecules into the membrane explains the experimental findings. The authors conclude that membrane permeability increases at the places where two or more antibiotic molecules form aggregates in the membrane.     

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.