Mode of binding of adriamycin with sulfatide-containing liposomes

Association constants and maximum binding numbers of sulfatide- and phosphatidylserine (PS)-containing liposomes for their binding with adriamycin (ADM) were determined by the column equilibration method. Although these liposomes have two kinds of binding sites, hydrophobic and electrostatic, the majority of ADM molecules was associated with the liposomes via their latter binding sites. Since the association constant for the electrostatic binding of ADM was smaller in sulfatide-containing liposomes (3 x 10(3) M-1) than in PS-containing ones (1.1 x 10(4) M-1), high efficiency of sulfatide-containing liposomes for the entrapment of ADM might be explained by the effect of sulfatide making the liposomes rigid, thereby preventing the leakage of the entrapped ADM.     

Construction of protocellular structures under simulated primitive earth conditions

We have developed experimental approaches for the construction of protocellular structures under simulated primitive earth conditions and studied their formation and characteristics. Three types of envelopes; protein envelopes, lipid envelopes, and lipid-protein envelopes are considered as candidates for protocellular structures. Simple protein envelopes and lipid envelopes are presumed to have originated at an early stage of chemical evolution, interaction mutually and then evolved into more complex envelopes composed of both lipids and proteins.Three kinds of protein envelopes were constructedin situ from amino acids under simulated primitive earth conditions such as a fresh water tide pool, a warm sea, and a submarine hydrothermal vent. One protein envelope was formed from a mixture of amino acid amides at 80 °C using multiple hydration-dehydration cycles. Marigranules, protein envelope structures, were produced from mixtures of glycine and acidic, basic and aromatic amino acids at 105 °C in a modified sea medium enriched with essential transition elements. Thermostable microspheres were also formed from a mixture of glycine, alanine, valine, and aspartic acid at 250 °C and above. The microspheres did not form at lower temperatures and consist of silicates and peptide-like polymers containing imide bonds and amino acid residues enriched in valine. Amphiphilic proteins with molecular weights of 2000 were necessary for the formation of the protein envelopes.Stable lipid envelopes were formed from different dialkyl phospholipids and fatty acids.Large, stable, lipid-protein envelopes were formed from egg lecithin and the solubilized marigranules. Polycations such as polylysine and polyhistidine, or basic proteins such as lysozyme and cytochromec also stabilized lipid-protein envelopes.     

Complete nucleotide sequence of the mitochondrial DNA from a liverwort,Marchantia polymorpha

Libraries of cosmid and plasmid clones covering the entire region of mtDNA from the liverwortMarchantia polymorpha were constructed. These clones were used for the determination of the complete nucleotide sequence of the liverwort mtDNA totally 186,608 bp (GenBank no. M68929) and including genes for 3 species of ribosomal RNAs, 29 genes for 27 species of transfer RNAs, and 30 genes for functionally known proteins (16 ribosomal proteins, 3 subunits of cytochromec oxidase, apocytochromeb protein, 3 subunits of H⁺-ATPase, and 7 subunits of NADH ubiquinone oxidoreductase). The genome also contains 32 unidentified open reading frames. Thus the complete nucleotide sequences from both chloroplast and mitochondrial genomes have been determined in the same organism. Plasmid clones are available upon the request. Gene names are represented according to Lonsdale and Leaver (1988) with modifications recommended by Lonsdale (personal communication).     

In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit.

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.     

A novel and efficient method for enzymatic synthesis of high purity maltose using moranoline (1-deoxynojirimycin).

A transglycosylation reaction with moranoline (1-deoxynojirimycin) was done with soluble starch as the glucosyl donor and Bacillus macerans amylase as a cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products with moranoline, obtained by treating the reaction mixture with a strong cation exchange resin, were hydrolyzed by beta-amylase [EC 3.2.1.2] from sweet potatoes. The hydrolysate was treated with a strong cation exchange resin, and high purity maltose was obtained.     

Cloning, Nucleotide Sequences and Differential Expression of the nifH and nifH-Like (frxC) Genes from the Filamentous Nitrogen-Fixing Cyanobacterium Plectonema boryanum

The frxC gene, found in liverwort chloroplast DNA, encodes aprotein of unknown function. The deduced amino acid sequenceof the protein shows significant homology to that of ni-trogenaseFe-protein encoded by the nifH gene. We have cloned the frxCand nifH genes from the nitrogen-fixing cyanobacterium Plectonemaboryanum, using frxC- and nifH-specific probes, and have determinedtheir nucleotide sequences. The amino acid sequence deducedfrom the frxC gene of P. boryanum exhibits 83% homology to thatof the protein encoded by the/rxCgene from liverwort, whereasit exhibits only 34% homology to that encoded by the nifH genefrom the same organism, namely, P. boryanum. Northern blot analysisshowed that the frxC gene was transcribed more actively undernitrogenase-repressed conditions than under nitrogenase-inducedconditions, suggesting that the FrxC protein has a functiondistinct from nitrogen fixation. These results, together withthe phylogenetic relationship between the nifH and frxC genes,indicate that the frxC and nifH genes are derived from a commonancestral gene but have evolved independently to encode proteinswith different functions. (Received April 27, 1991; Accepted August 12, 1991)     

Distribution and characterization of diglycosyldiacylglycerol isomers with different anomeric configuration in higher plants.

The novel diglycosyldiacylglycerol (DGDG), galactosyl(beta 1----6)-galactosyl(beta 1----3')-diacylglycerol (B isomer), present in Adzuki beans was found to be distributed together with the well-known galactosyl(alpha 1----6)-galactosyl(beta 1----3')-diacylglycerol (A isomer), in all (10) of the higher plants examined. The highest levels were found in leguminous seeds were the amounts were always less than 33% of the total DGDG of mature seeds. The highest proportion of the B isomer was found in Adzuki bean seed DGDG (26-33%), with the lowest in pea seed DGDG (2%). The amounts of the B isomer in DGDG of Adzuki and kidney beans cotyledons were almost equal to those in mature seeds. Immature seeds and hypocotyls of three kinds of beans also contained the B isomer in small amounts compared with the mature beans, while only trace amounts of the isomer was found in other organs such as leaves, stems, pods, roots and generative organs of plants, except for root from kidney beans. The molecular species composition of the principal diacylglycerol moieties in the A and B isomers of DGDG were found to be significantly different among several plant seeds, although the component diacylglycerol species were qualitatively similar to each other.     

Novel Technique for Measuring Tissue Firmness within Tomato (Lycopersicon esculentum Mill.) Fruit

Developmental changes of tomato (Lycopersicon esculentum) fruit tissues during maturation were analyzed by a physically defined method (stress-relaxation analysis). The tip of a conical probe connected to a load sensor was positioned on the cut surface of a sliced tomato fruit, and the decay of the imposed stress was monitored. Stress-relaxation data thus obtained were used for the calculation of three stress-relaxation parameters. Different zones within tomato fruit harvested at six different ripening stages were analyzed. One of the stress-relaxation parameters, minimum stress-relaxation time (T₀), decreased as the fruits matured. The decrease in T₀ was first found in the core of the carpel junction within the endopericarp at the blossom end during the breaker stage. The decrease in T₀ progressed from the blossom end, through the equatorial region and finally throughout the shoulder, as the fruit matured. In mature green fruit, T₀ values within the placenta and the proximal carpel junction were lower than those by other parts of the fruit. For all measurements the maximum stress-relaxation time was not substantially changed during maturation, nor were their changes observed in different regions of the fruit. The observed relaxation rate was therefore correlated with softening. The results indicate that fruit softening may be physically associated with the stress-relaxation parameter, T₀, and the extent of softening is a function of position within the fruit. Decreases in T₀ value appear to be correlated with the reported regional variation in the appearance of polygalacturonase.     

Expression of sodium pump activities in BALB/c 3T3 cells transfected with cDNA encoding alpha 3-subunits of rat brain Na+,K+-ATPase

The cDNAs encoding alpha 3-subunits of rat brain Na+,K+-ATPase and the neomycin resistance gene were incorporated into BALB/c 3T3 cells by the co-transfection method. Stably transformed cells were selected with 300 micrograms/ml of neomycin (G-418) for 6 weeks. Northern blot analysis using the 3'-non-translated region of the cDNA as a probe revealed that the alpha 3 mRNA appeared in transfected cells. Na+,K+-ATPase activity of the transfected cells was twice that of wild-type cells. Regarding ouabain sensitivity, the Na+,K+-ATPase showed two Ki values for ouabain (8 x 10(-8) and 4.5 x 10(-5) M) in transfected cells while wild-type cells displayed only the higher value. Ouabain sensitivity of Rb+ uptake also demonstrated two Ki values in the transfected cells (8 x 10(-8) and 4 x 10(-5) M) and a Ki in wild-type cells of 4 x 10(-5) M. It is concluded that alpha 3 is a highly ouabain-sensitive catalytic subunit of Na+,K+-ATPase. It is also suggested that ouabain sensitivity is exclusively determined by the properties of the alpha-subunit rather than the beta-subunit. This is the first report on the catalytic characteristics of the alpha 3 isoform of Na+,K+-ATPase.