Cryopreservation of spermatozoa of a transgenic mouse]

Spermatozoa of a homozygous transgenic mouse, in which the firefly luciferase gene was expressed under the control of beta-actin promoter, were frozen at -196 degrees C. One fourth of the frozen sperm was later thawed and used for in vitro fertilization. Thirty-six of 65 oocytes (55.4%) developed to the 2-cell stage. All the 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 23 young (63.9%, 23/36) were born. All of young analyzed carried the transgene and showed the luciferase gene expression.     

Transgenic tobacco resistant to a bacterial disease by the detoxification of a pathogenic toxin

Summary Some plant pathogens produce toxins which cause disease in infected plants. One of the pathogenic toxins, tabtoxin, is produced by Pseudomonas syringae pv. tabaci, which causes wildfire of tobacco. A tabtoxin resistance gene (ttr) coding for an acetyltransferase isolated from Pseudomonas syringae pv. tabaci was fused to the 35S promoter of the cauliflower mosaic virus (CaMV) to construct a chimeric gene for introduction into tobacco cells by Agrobacterium-mediated transformation. The transgenic tobacco plants showed high specific-expression of the ttr gene and no chlorotic symptoms caused by tabtoxin treatment or with infection by Pseudomonas syringae pv. tabaci. These results demonstrate a successful approach to obtain disease-resistant plants by detoxification of the pathogenic toxins which play an important role in pathogenesis.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and Sequencing of a Genomic Clone for Mouse Brain Specific Small RNA

We isolated a mouse genomic clone that hybridized with small RNA present in the cytoplasm of the brain. The RNA was about 150 nucleotides long. This RNA seemed to be specific to the brain, since it was not found in the liver or kidney. The clone DNA contained a sequence homologous to 82-nucleotide "identifier" core sequence of cDNA clones of rat. The sequence contained a split promoter for RNA polymerase III and was flanked by a 12-nucleotide direct repeat (ATAAATAATTTA).     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.     

Induction of berberine biosynthesis by cytokinins in Thalictrum minus cell suspension cultures

Production of berberine could be induced by adding 6-benzylaminopurine (BAP) to Thalictrum minus cells, cultured in suspension in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), early in the growth cycle. In the presence of BAP, the precursor, L-tyrosine, was rapidly converted into berberine which was then released into the medium, whereas substantial amounts of the intermediates, tyramine and dopamine, accumulated in non-berberine-producing cells grown in the same 2,4-D-containing medium without BAP. These results suggest that BAP activated enzymatic reactions subsequent to the formation of the amines in the biosynthesis of berberine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAP 6-isopentenylaminopurine - LS medium Linsmaier-Skoog medium - Growth medium LS medium containing 10^-6 M 2,4-D     

Effects of alginates on the ingestion and excretion of cholesterol in the rat

The effects of three forms of alginate (free acid, sodium and calcium salts) on ingestion and excretion of cholesterol in the rat were studied. For two weeks, Male Sprague-Dawley rats were fed a cholesterol-rich diet containing 3% alginate from the brown alga Ascophyllum nodosum. The food efficiency of the three types of alginate was: Na-alginate > Ca-alginate > alginic acid. A significant increase in the weight of cecum was also observed in alginate diets. Alginate was not effective in preventing the elevation of serum total cholesterol levels, although irregularly changing patterns were observed. The cholesterol level of liver showed a tendency to decrease with alginate feeding, while fecal excretion of cholesterol increased.