Glycerol permeability of human spermatozoa and its activation energy

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.     

Three-dimensional solution structure of apo-neocarzinostatin.

Two and three-dimensional solution nuclear magnetic resonance studies of the 11K apoprotein from natural antitumor agent neocarzinostatin (NCS) were extended to elucidation of the high-resolution structure by the use of restrained molecular dynamics computations. The refined structures attained convergency upon three steps of iterative calculations, in which more distance restraints were extracted from experimental data, and the existing distance bounds were optimized on the basis of computed structures. The solution structures of apo-NCS contain seven antiparallel beta-strands, which form two closely located beta-sheets and a short beta-segment. This protein lacks any alpha-helical component. The alignment of the seven beta-strands gives rise to a beta-barrel with an elongated diameter in one direction. The global structure of apo-NCS resembles that of the Ig-fold domain found in immunoglobulins and other structurally related beta-proteins. Residues responsible for side-chain packing and the possible salt-bridge formation important for protein folding were identified. Neocarzinostatin and the analogous proteins are known to exert their biological activity through the interaction of DNA with a chromophoric molecule, which is non-covalently bound to the apo-proteins. This molecular chromophore-binding site in apo-NCS is made of a cavity consisting of residues from the four-beta-stranded sheet and the short beta-segment. Although the solution structures of apo-NCS are similar to that of the analogous apoauromomycin in the crystalline state, difference in the shape of the binding cavities between the two was found. This study provides a structural basis for characterization of the specific recognition and molecular mechanism of the antitumor NCS chromophore binding to its host protein.     

Genotoxicity of heated cooking oil vapors.

Epidemiological studies of lung cancer in Chinese women indicated that factors other than cigarette smoking are related to lung cancer risk. A case-control study suggested that indoor air pollution, particularly from cooking oil emissions, may be involved. Condensates of volatile emissions from rapeseed and soybean cooking oils were prepared and found to be genotoxic in short-term tests including the Salmonella mutation assay, SV50 forward-mutation assay, and sister-chromatid exchange assay, as well as the micronucleus assay in mouse bone marrow. In contrast, condensates from rapeseed oil with butylated hydroxyanisole or hydrogenated rapeseed oil were not mutagenic, implicating oxidation products as the cause for mutagenicity. Peanut oil and lard condensates were not mutagenic in any assay. The association of exposure to Chinese rapeseed cooking-oil emissions and lung-cancer risk may be related to the mutagenic component of these condensates.     

A cytoplasmic chaperonin that catalyzes beta-actin folding.

We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin. The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity. The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product. Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP. The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts.     

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxidase-containing proteoliposomes.

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.     

The stability of foreign protein production in genetically modified plant cells

Summary The stability of foreign protein production in genetically engineered plant cells was studied. A cultured tobacco cell line was transformed with a chimeric molecule carrying a bacterial gene, ß-glucuronidase (GUS), under plant regulatory sequences. The specific GUS activity was monitored for 294 days with ten independently transformed cell lines either in the presence or the absence of selectable antibiotics. Specific GUS activity was stably maintained in five lines. About a two-to four-fold increase in the GUS activity was observed from three cell lines. The remaining two cell lines lost the activity within the first 70 to 210 days. The presence of antibiotics did not significantly alter the stability of the foreign protein production in all cell lines examined.     

Molecular structure of antitumor drug steffimycin and modelling of its binding to DNA.

The molecular and crystal structure of steffimycin have been determined by single crystal X-ray diffraction to 0.9 angstrom resolution. The triclinic crystals are in the space group P1, with the unit cell dimensions of a = 8.606(3) angstrom, b = 22.168(7) angstrom, c = 8.448(2) angstrom, alpha = 97.56(3) degrees, beta = 95.97(2) degrees, gamma = 87.94(3) degrees, Z = 2. The structure was solved by direct methods and refined by the full-matrix least-squares method to a final R value of 0.065 with 3405 (Inet greater than 2.0 sigma (Inet] observed reflections using the NRCVAX software package. The crystal lattice includes 2 independent steffimycin, 3 water and one 2-methyl-2,4-pentanediol molecules. The conformation of steffimycin is grossly similar to other anthracycline antibiotics including daunorubicin. The crystal packing interactions of steffimycin suggest a preferred stacking of the aglycone chromophore of the antibiotic which resembles the intercalative interactions seen in the daunorubicin-d(CGTACG) (Wang et al., Biochemistry 26, 1152 (1987] and nogalamycin-d(CGT(pS)ACG) (Liaw et al., Biochemistry 28, 9913 (1989] complexes. The atomic coordinates data from these complexes were used to model the intercalative binding of steffimycin to DNA. The models were then stereochemically idealized by the constraint refinement program NUCLSQ. Subsequently XPLOR software package was used for energy minimization of these models in vacuo. The model building studies suggest that steffimycin has a higher CpG base sequence specificity over the TpA step, similar to that of daunorubicin and nogalamycin.     

Enhanced growth of the red algaPorphyra yezoensis Ueda in high CO2 concentrations

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO₂. It was found that the higher the CO₂ concentration, the faster the growth of the thalli. Aeration with elevated CO₂ lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO ₃ ^? to OH^? and CO₂ proceeded much faster than the dissociation of hydrated CO₂ releasing H⁺. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO₂ + HCO ₃ ^? + CO ₃ ^? ). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO₂ by marine algae is discussed.