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.     

Chromosomal localization of the human annexin III (ANX3) gene

The annexins or lipocortins are a new family of calcium-dependent phospholipid-binding proteins. Annexin III has been previously identified as inositol 1,2-cyclic phosphate 2-phosphohydrolase (EC 3.1.4.36), an enzyme of inositol phosphate metabolism, and also as placental anticoagulant protein III, lipocortin III, calcimedin 35-alpha, and an abundant neutrophil cytoplasmic protein. In this study, the gene (ANX3) encoding annexin III was localized to human chromosome 4 at band q21 (q13-q22) by (1) polymerase chain reaction analysis of a human-rodent hybrid cell panel, confirmed by genomic Southern blot analysis of the same panel with a cDNA probe and (2) in situ hybridization with a cDNA probe.     

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.     

人αA干扰素在克鲁氏乳酸酵母中的表达和分泌

pE1是由酵母天然质粒pKD1衍生出来的重组穿梭质粒,在克鲁氏乳酸酵母中具有高拷贝,高稳定性等特点。把人αA干扰素分泌表达单元克隆到pE1载体中,得到分泌型表达质粒pE-IFN1。pE-IFN1在克鲁氏乳酸酵母中相当稳定,在非选择性培养基中生长50世代后,大多数酵母细胞仍带有质粒。结果表明,分泌表达单元中的酿酒酵母α因子的分泌信号肽能被克鲁氏乳酸酵母的蛋白质分泌系统所识别,人αA干扰素被分泌到细胞外。在摇瓶培养条件下每升发酵液中含有1—2毫克αA干扰素。     

Effect of oxygen supply on the suspension culture of genetically modified tobacco cells.

The effect of oxygen supply on the cultivation of the genetically modified tobacco cells and the formation of a foreign protein, beta-glucuronidase (GUS), was investigated in 250-mL Erlenmeyer flasks, a 5-L stirred tank fermenter, and a 7-L air-lift fermenter. The oxygen supply was varied by using different volumes of medium in the case of the 250-mL Erlenmeyer flask culture or by the different aeration rate in the case of the two types of fermenters tested. Higher oxygen supply stimulated cell growth and increased oxygen consumption rate, the level of phenolics, and GUS productions.