Standardized extraction method for paralytic shellfish toxins in phytoplankton

The optimal conditions were established for extraction of paralytic shellfish toxins from a Danish clone of Alexandrium tamarense using extraction with acetic acid and HCl in the concentration range 0.01–1.0 N. Physical destruction of the cells was investigated microscopically to select the most efficient extraction procedure.The toxin content was quantitated by an automized isocratic reversed-phase high-performance liquid chromatography (HPLC) method. The best results as judged from the total amount of toxins and the toxin profile were obtained using 0.05–1.0 N acetic acid and 0.01–0.02 N HCl. Hydrochloric acid in the concentration range 0.03–1.0 N caused the amount of C1 and C2 toxins to decrease sharply and concomitant increase of gonyautoxins 2 and 3.The phytoplankton extracts with 0.1 to 0.5 N acetic acid or 0.01 N HCl were stable during 6 months at –20 °C, but the extracts with HCl 0.02 N underwent a change in toxin profile, although the total amount of toxins was constant.     

Clonal tumorigenic endodermal cell lines producing basement membrane components

Tumors were developed from the mouse teratocarcinoma-derived endodermal cell line PF HR-9 by subcutaneous injections in syngeneic mice of large numbers of cells previously cultured for several years at high cell density. Cell cultures were established from the tumors and the cells were cloned. The cloned sublines were highly malignant in vivo and tumor metastases were occasionally observed. The tumors contained abundant extracellular material, which was distinctly laminated and contained type IV collagen, laminin, and heparan sulfate proteoglycan. The tumorigenic sublines were also shown to have retained markers of the original cells, such as the cytoskeletal proteins Endo A and B. These cell lines should be useful for biosynthetic studies on basement membrane and cytoskeletal components and the tumors for isolation of these macromolecules and their mRNAs.     

Localization of triphosphoinositide in the cochlea

Summary Triphosphoinositide (TPI) has been demonstrated to be a receptor for aminoglycosides in the cochlea and may regulate ionic permeability by its binding with Ca⁺⁺. This phospholipid was localized by a protein A-gold technique in the cochlea at the electronmicroscopic level. TPI was prepared by a neomycin column and antibodies to it were raised in rabbits. The antibody used in this study reacted virtually only to TPI among the tested lipids. TPI was localized mainly at stereocilia, cuticular plates, head plates of Deiter's cells, plasma membrane, and mitochondria of various cells in the organ of Corti. In the vascular stria, TPI was found mainly at the plasma membrane of basal infoldings of the marginal cells. Possible physiological and pathophysiological roles of TPI in the cochlea are briefly discussed.     

Frequencies of Twelve Ascus-Types and Arrangement of Three Genes from Tetrad Data

Equations expressing the theoretical frequencies of twelve ascus-types in the tetrad analysis of a triply heterozygous diploid are described. Using these equations, a mapping procedure for a gene X, is proposed. The procedure requires that two genes, X and Y, of the same phenotype be heterozygous and that the map position of Y be known, and that another standard gene, Z, show an independent phenotype from X and Y. This procedure does not require the laborious allelism test of the segregants to determine the allelic 2:2 segregation in tetrads for the X and Y genes, which is indispensable for mapping by the conventional procedure. The exact placement of the X gene on a chromosome is possible by the chi2 minimization procedure in comparison with the expected frequencies of the six ascus-types or four spore-types deduced from the twelve expected ascus-types to give the optimal fit with the observed data.     

Proton magnetic resonance spectra of tRNA-Met-f from Thermus thermophilus.

220 MHz proton magnetic resonance spectra of tRNAs in bulk and tRNA-Met-f from Thermus thermophilus have been measured and compared with those of tRNAs from E. coli. Temperature dependences and chemical shift positions of the bulk tRNAs are well explained by the difference in their GC contents. It is known that the base sequence of the double helical regions in the cloverleaf structure of T. thermophilus tRNA-Met-f is different from that of E. coli tRNA-Met-f only at two positions in TpsiCarm; one more C:G pair is contained instead of a U:G pair of E. coli tRNA-Met-f and a C:G pair of E. coli is replaced by a G:C pair. In spite of the resembrance in the base sequences, nmr patterns around 13 ppm are fairly different from each other. The difference is discussed in relation with their tertiary structures and with the origin of chemical shift displacements.     

C-type cytochromes isloated from an extreme thermophile, Thermus thermophilus HB8.

Two cytochromes of the C-type, c-554 and c-549, were isolated from the soluble fraction of an extreme thermophile, Thermus thermophilus HB8. Highly purified cytochrome c-554 had absorption maxima at 554, 522, and 417 nm in the reduced state, and at 410 nm in the oxidized state. The alpha-band of the reduced state resembled that of "split-alpha" cytochromes. The isoelectric point was at pH 4.9, and the molecular weight was about 29,000. Cytochrome c-549, partially purified, had absorption maxima a6 549,520, and 416 nm in the reduced form, and at 408 nm in the oxidized form. The molecular weight was about 25,000. Both were slowly auto-oxidizable, and did not combine with CO.     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile, Thermus aquaticus YT-1

A gene (leuB) coding for 3-isopropylmalate dehydrogenase [EC 1.1.1.85] from an extreme thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined. It contains an open reading frame of 1,035 bp encoding 344 amino acid residues. The homology with that from T. thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences. No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB [Croft et al. (1987) Mol. Gen. Genet. 210, 490-497]. Substitutions in the primary structure which are unique for the thermophile sequences are discussed in relation to the unusual stability of the thermophile dehydrogenase based on amino acid sequence comparison of 9 microorganisms including thermophiles and mesophiles.