Immunological detection of propolypeptide of von Willebrand factor on platelet surface

Recently we have found that propolypeptide of von Willebrand factor (pp-vWF) obtained from platelets binds to type I collagen. It is known that pp-vWF is present in platelet alpha-granules and is secreted upon activation. In this paper, we demonstrate the two following evidences to show that it is also present on the surface of resting platelets. [1] The antibody against pp-vWF bound to the surface of platelets. [2] The antibody induced aggregation of platelets. The binding of the antibody and the antibody-induced aggregation of platelets were inhibited in a dose-dependent manner by Fab fragment of the antibody. Platelets from von Willebrand disease patients bound less of the antibody and responded weakly to the antibody.     

Primary structure of two linker chains of the extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus

Two types of linker subunits (linkers 1 and 2) of the extracellular hemoglobin of Tylorrhynchus heterochaetus have been isolated as disulfide-linked homodimers by C18 reverse-phase chromatography. These subunits constituted 6 and 13%, respectively, of total protein area on the chromatogram. The complete amino acid sequences of linkers 1 and 2 were determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, pepsin, and endoproteinase Asp-N. The linker 1 consisted of 253 amino acid residues (the calculated molecular mass, 28,200 Da), while the linker 2 consisted of 236 residues (26,316 Da). The two chains showed 27% sequence identity. The amino acid sequences of Tylorrhynchus linkers 1 and 2 also showed 23-27% homology with the recently determined sequence of a linker chain of Lamellibrachia hemoglobin (Suzuki, T., Takagi, T., and Ohta, S. (1990) J. Biol. Chem. 265, 1551-1555). In the three linker chains, half-cystine residues were highly conserved; 8 out of 13 residues are identical, suggesting that such residues would contribute to the formation of intrachain disulfide bonds essential for the protein folding of the linker polypeptides. Based on the exact molecular masses of the linker and the heme-containing subunits, the molar ratios estimated for the subunits and the minimum molecular weights per 1 mol of heme, a model is proposed for the subunit structure of the Tylorrhynchus hemoglobin, consisting of 216 polypeptide chains, 192 heme-containing chains, and 24 linker chains.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.     

Nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions.

The nucleotide sequence of Physarum polycephalum 5.8S rRNA gene and its flanking regions has been determined. The homologies of the 5.8S rRNA sequence with those of Saccharomyces, Chlamydomonas and Xenopus were 56%, 50% and 52%, respectively. In spite of these relatively low homologies, its possible secondary structure was very similar to those of other species.     

The late-replicating X chromosome in digynous mouse triploid embryos

Using BrdU-labeling and acridine orange staining, the behavior of X-chromosome replication was studied in 28 XXX and 19 XXY digynous mouse triploids. In some of these the paternal and maternal X chromosome could by cytologically distinguished. Such embryos were obtained by mating chromosomally normal females with males carrying Cattanach's X chromosome which contains an autosomal insertion that substantially increases the length of this chromosome. In the XXX triploids there were two distinct cell lines, one with two late-replicating X chromosomes, and the other with only one late-replicating X. The XXY triploids were also composed of two cell populations, one with a single late-replicating X and the other with no late replicating X chromosome. Assuming that the late-replicating X is genetically inactive, in both XXX and XXY triploids, cells from the embryonic region tended to have only one active X chromosome, whereas those from the extra-embryonic membranes tended to have two active X chromosomes. The single active X chromosome was either paternal or maternal in origin, but two active X chromosomes were overwhelmingly maternal in origin, suggesting paternal X-inactivation in extra-embryonic tissues.     

Two cage-type lupin alkaloids from Sophora franchetiana

Two new cage-type lupin alkaloids, (?)-tsukushinamine-B and tsukushinamine-C, have been isolated from the fresh epigeal parts of Sophora franchetiana, along with (?)-cytisine, (?)-N-formylcytisine, (?)-rhombifoline, (?)-anagyrine, (?)-baptifoline and (±)-ammodendrine, as well as (?)-tsukushinamine-A. The structures of these novel tsukushinamine-type lupin alkaloids were determined by spectroscopic data and partly by a chemical reaction. Variations of the alkaloid contents in the seeds, seedlings and various parts of S. franchetiana were also examined.     

Study of oxytocin receptor: II. Gestational changes in oxytocin activity in the human myometrium

This study was designed to investigate the oxytocin (OT) specific binding receptors in 20,000 x g pellets of nonpregnant, first trimester and term human myometria. The receptor analysis was done using the lower uterine segment at term and the lower portion of the anterior uterine body in nonpregnant and first trimester subjects, and no difference was found in the myometrial receptor concentrations in the various uteri. The mean +/- S.D. values of the receptor dissociation constants were 3.33 +/- 0.50, 2.71 +/- 1.03 and 1.87 +/- 0.30 nM and the number of binding sites was 0.30 +/- 0.10, 0.50 +/- 0.10 and 1.50 +/- 0.50 pmol/mg protein at each stage studied, indicating that the gestational increase of uterine sensitivity to OT is due to the increase in myometrial OT binding sites as well as its binding affinity. Further, myometrial OT binding before and after the onset of labor was studied and a marked decrease in total myometrial OT binding was noticed; 35.6 +/- 13.0% before and 20.2 +/- 5.0% after. This decrease was thought to be due to the decrease in the number of binding sites from 1.50 +/- 0.50 to 0.74 +/- 0.21 pmol/mg protein after the onset of labor (p less than 0.01). No changes were found in the dissociation constants. Thus it seems that OT and its receptor coupling triggers labor or is involved in the early steps of labor.     

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.     

Plasma levels of 16 alpha-hydroxypregnenolone, 16 alpha-hydroxyprogesterone and 16 alpha-hydroxydehydroepiandrosterone in the fetus and neonates.

Simultaneous determination of unconjugated 16 alpha-hydroxypregnenolone (16 alphaOH-Preg), 16 alpha-hydroxyprogesterone (16 alphaOH-Prog) and 16 alpha-hydroxydehydroepiandrosterone (16 alphaOH-DHEA) in fetal and neonatal plasma was performed utilizing a newly developed radioimmunoassay. In all neonates, the three 16 alpha-hydroxysteroid levels were consistently higher in umbilical cord plasma than in the maternal peripheral circulation. 16 alpha-OH-Preg in the umbilical arterial plasma increased from 11.2 +/- 3.1 at 24 weeks to 29.7 +/- 12.0 ng/ml at term, 16 alphaOH-Prog from 15.5 +/- 3.2 to 34.3 +/- 11.0 ng/ml and 16 alphaOH-DHEA from 5.1 +/- 1.2 to 5.9 +/- 1.0 ng/ml. In the anencephalic neonates, only 16 alphaOH-Preg showed an increase pattern under ACTH priming. 16 alpha-OH-Preg levels for normal full term neonates remain relatively constant at the first 24 hr and show a slight decrease at 3 days post partum. In small full term neonates, 16 alphaOH-Preg levels in umbilical arterial plasma are considerably higher than in normal neonates and remain at roughly equivalent levels for the first 5 days post partum. 16 alphaOH-Prog and 16 alphaOH-DHEA levels in umbilical arterial plasma in normal and small full term neonates are almost equal and both groups show a rapid decrease during the first 24 hr. Comparison with findings of the three 16 alpha-hydroxysteroids in fetal and neonatal plasma is discussed.