Photochemical cycloaddition of 1,3-diacetoxy-2-propanone to (trimethylsilyloxy)ethylene

The structures of two capsular polysaccharides elaborated by Haemophilus influenzae type e, strains NCTC 8455 and 8472, respectively, have been investigated, methylation analysis and n.m.r. spectrometry being the principal methods used. It is concluded that the polysaccharides are composed of repeating-units having the following structure:

In the polysaccharide from strain NCTC 8472, all of the repeating-units contain the β-dfructofuranosyl group. The polysaccharide from strain NCTC 8455, however, contains only traces of d-fructose, corresponding to approximately one group per 100 repeating-units.     

Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.     

Immunolocalization of a 25-kilodalton protein in mouse testis and epididymis.

We have recently observed that a polyclonal antibody raised against a mouse epididymal luminal fluid protein (MEP 9) recognizes a 25-kDa antigen in mouse testis and epididymis [Rankin et al., Biol Reprod 1992; 46:747-766]. This antigen was localized by light and electron microscopic immunohistochemistry. The immunoreactivity in the testis was found in the residual cytoplasm of the elongated spermatids, in the residual bodies, and in the cytoplasmic droplets of spermatozoa. In the epididymis, the epithelial principal cells were stained from the distal caput to the distal cauda. Immunogold labeling in the principal cells showed diffuse distribution without preferential accumulation in either the endocytic or the secretory apparatus of the cells. In the epididymal lumen, the immunoreactivity was restricted to the sperm cytoplasmic droplets. No membrane-specific labeling was observed in luminal spermatozoa, cytoplasmic droplets, or isolated sperm plasma membranes. Three weeks after hemicastration or severance of the efferent ducts, a normal distribution of the immunoreactive sites was found in the epididymis. Immunoreactivity, was also detected in the epididymal epithelium of immature mice as well as in that of XXSxr male mice having no spermatozoa in the epididymis. These results suggest that the immunoreactivity seen in the principal cells originates from synthesis rather than endocytosis of the testicular protein from disrupted cytoplasmic droplets. Furthermore, these results suggest that the 25-kDa protein is synthesized independently by both testis and epididymis.     

The distribution of diploids and polyploids of theScilla scilloides complex in the northeastern district of China

A cytological analysis was made of 1504 individuals ofScilla scilloides Druce from the northeastern district of China. In terms of genome combination four cytotypes were found: AA (90.95%), AAAA (0.07%), AABB (8.92%) and AABB+2 (0.07%). In the cytotypes A and B denote genome A (x=8) and genome B (x=9), respectively. Most of 18 natural populations examined consist of one cytotype: indeed, all individuals examined of 15 of the populations are AA diploids, those of one population AABB allotetraploids, and individuals examined of two other populations have two cytotypes each, AA and AAAA, and AABB and AABB+2. In karyotype the genome A of the AA and AAAA was different from that of the AABB, and a direction of morphological change of chromosomes in the genome A is discussed in light of results of hybridization experiments reported elsewhere. Taken together with information available for the distribution of different cytotypes in China, Korea and Japan, the results of the present study support the view of Maekawa thatScilla scilloides was native to mainland China and lately introduced to Japan.     

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.     

Electron microscopy of histamine-induced contraction of the in vitro swine coronary artery.

Dose-dependent contractions of the in vitro swine coronary artery were induced by application of histamine and acetylcholine, but not of angiotensin II, ergonovine, noradrenaline, prostaglandin F2 alpha and serotonin. Ultrastructural changes especially of the tunica intima during the contractions were observed at 2, 5 and 30 min after application of histamine and acetylcholine. The intimal gutter spirally running along the longitudinal axis of the vessel was obscured, and the intimal surface became extensively indented. Exclusively in the histamine-treated samples, the increase in number and size of the intracellular vacuoles and the dilation of the intercellular clefts to the extent of the intercellular vacuoles were observed in the endothelium. Moreover, the enhancement of the endothelial permeability was indicated by the marker experiments using horseradish peroxidase. Such endothelial cell damages and the enhancement of the endothelial permeability may amplify the coronary artery contraction.     

Primary structure of Vicia angustifolia proteinase inhibitor

The complete amino acid sequence (72 amino acid residues) of a double-headed proteinase inhibitor from seeds of Vicia angustifolia L. var. segetalis Koch has been determined and compared with those of other double-headed inhibitors of known structure. Sequencing was performed by conventional methods with the aid of the fragments produced by reduction and S-carboxymethylation of the enzymatically modified inhibitors, and also using tryptic and chymotryptic peptides. The positions of the 14 half-cystine residues agreed among all the reported primary structures of the legume double-headed inhibitors. However, V. angustifolia inhibitor possessed extensive amino acid differences compared to the others. The phylogenetic relationship among these inhibitors was established using the unweighted pair-group method and revealed that the V. angustifolia inhibitor and the peanut inhibitor B-III had diverged at a relatively earlier stage compared to the other inhibitors.     

Tissue-specific and developmental expression of human transthyretin gene in transgenic mice

To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.