Identification of viruses infected in Rehmannia glutinosa Libosch. var purpurea Makino and effect of virus infection on root yield and iridoid glycoside contents

Virus free plants of Rehmannia glutinosa Libosch. var. purpurea Makino were obtained through meristem tip tissue cultures from plants infected with a mixture of tabocco mosaic virus(TMV), a member of the carlavirus group, and an unknown spherical virus. The re-infection rate of the virus free plants by TMV in the field was determined by enzyme linked immunosorbent assay(ELISA). Twenty seven percent of the plants were re-infected during the first year, 31 % by the end of second year, and 63 % by the end of the third year. The yield of root and iridoid glycoside contents gradually decreased each year. These results led to the conclusion that virus infection causes marked decrease of the yield of roots and productivity of secondary metabolites.     

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.     

Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of α and β subunits and has a tetra-chain arrangement (β-α-α-β) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two αβ units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the α chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two α chains. This was confirmed by amino acid sequence analysis of the α chains: that is, Cys₁₅ participating in the inter-α chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, α chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their α chains and were not glycosylated.     

Phylogenetic relationships among eutherian orders estimated from inferred sequences of mitochondrial proteins: Instability of a tree based on a single gene

The phylogenetic relationships among Primates (human), Artiodactyla (cow), Cetacea (whale), Carnivora (seal), and Rodentia (mouse and rat) were estimated from the inferred amino acid sequences of the mitochondrial genomes using Marsupialia (opossum), Aves (chicken), and Amphibia (Xenopus) as an outgroup. The overall evidence of the maximum likelihood analysis suggests that Rodentia is an outgroup to the other four eutherian orders and that Cetacea and Artiodactyla form a clade with Carnivora as a sister taxon irrespective of the assumed model for amino acid substitutions. Although there remains an uncertainty concerning the relation among Artiodactyla, Cetacea, and Carnivora, the existence of a clade formed by these three orders and the outgroup status of Rodentia to the other eutherian orders seems to be firmly established. However, analyses of individual genes do not necessarily conform to this conclusion, and some of the genes reject the putatively correct tree with nearly 5% significance. Although this discrepancy can be due to convergent or parallel evolution in the specific genes, it was pointed out that, even without a particular reason, such a discrepancy can occur in 5% of the cases if the branching among the orders in question occurred within a short period. Due to uncertainty about the assumed model underlying the phylogenetic inference, this can occur even more frequently. This demonstrates the importance of analyzing enough sequences to avoid the danger of concluding an erroneous tree.