Visualization method for sulfolipids and its application to the determination of nanomole quantities of lipid sulfur

A simple and sensitive visualization method for sulfolipids on a thin-layer chromatogram is described. By spraying with an acidic solution of azure A, a complex was formed between an anionic sulfolipid and a blue cationic compound. After the unbound dye was washed out by brief soaking in methanol, sulfolipids were visualized as clear dark-blue bands on a light-blue background. As little as 0.5 nmol could be detected. Sulfolipid-dye complex was estimated by densitometry or colorimetric measurement after extraction with chloroform/methanol. For the quantitative determination of sulfolipids having long sugar chains, it is necessary to treat thin-layer chromatography plates with acetic anhydride before color development. Of the other tissue lipids not containing sulfuric acid ester that were tested none were stained significantly. A linearity of quantitative determination was observed over the range of 1-8 nmol.     

Mutations upstream of the ribosome-binding site affect translational efficiency

The DNA coding for the major outer membrane lipoprotein of Escherichia coli has been fused to the coding region of the beta-galactosidase gene to measure the effect of various mutations on the efficiency of translation initiation. The various mutants were made by either inserting or deleting a small number of nucleotides into or from a region just upstream of the ribosome-binding site. These small mutations dramatically affect translation initiation as measured by the production of beta-galactosidase. We postulate that these mutations affect translation initiation by altering the secondary structure of the messenger RNA. In one case, we predict that a stem and loop just upstream of the Shine-Dalgarno sequence sterically hinders the binding of the ribosome to the mRNA.     

Methyl cinnamate derivatives enhance UV-induced mutagenesis due to the inhibition of DNA excision repair in Escherichia coli B/r

UV-induced mutagenesis in Escherichia coli B/r WP2 was enhanced by certain derivatives of methyl cinnamate which themselves were not mutagenic. Methyl ferulate, methyl isoferulate and methyl sinapate showed this effect markedly. Such an enhancement effect was absent with the derivatives of cinnamic acid and ethyl cinnamate and was not observed in Escherichia coli WP2s uvrA. Methyl sinapate also enhanced 4NQO-induced mutation and suppressed liquid-holding recovery in the above repair-proficient strain. The presence of methyl sinapate in plating agar medium decreased the survival of UV-irradiated cells of a recombination-repair-deficient strain, CM571 recA. However, the effect was not observed with those of WP2s uvrA. In an in vitro experiment in which the removal rate of thymine dimers was measured, methyl sinapate clearly inhibited this repair event. From these results, we conclude that methyl sinapate inhibits DNA excision repair, thus enhancing UV mutagenicity.     

Paratropomyosin: a new myofibrillar protein that modifies the actin-myosin interaction in postrigor skeletal muscle. I. Preparation and characterization

A protein component which is released from skeletal-muscle myofibrils on the treatment with Ca2+ at concentrations above 10(-5) M and modifies the actin-myosin interaction was purified by a method involving column chromatography on Sephadex G-200 and DEAE-cellulose in succession. Although this protein resembles tropomyosin in some physicochemical properties, it has the same chain weight of 34,000 as the alpha-component of tropomyosin on SDS-polyacrylamide gel electrophoresis, and differed from tropomyosin not only in the amino acid composition but also in prolonging the clearing phase of superprecipitation of reconstituted actomyosin. We therefore concluded that this protein is a new myofibrillar one, and termed it "paratropomyosin." In postrigor muscle, it seems likely that paratropomyosin is released from its original locus with an increased concentration of Ca2+, and that it weakens rigor linkages formed between actin and myosin.     

Fibrinopeptides A and B of Japanese monkey (Macaca fuscata) and patas monkey (Erythrocebus patas): their amino acid sequences, restricted mutations, and a molecular phylogeny for macaques, guenons, and baboons

Amino acid sequences of fibrinopeptides A and B from the macaque, Macaca fuscata (Japanese monkey) and the guenon, Erythrocebus patas (patas monkey) were established. Fibrinopeptides A of the monkeys had a sequence identical with those of baboons: Ala-Asp-Thr-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg. Fibrinopeptides B were 9-residue, "short," peptides with the sequences Asn-Glu-Glu-Ser-Leu-Phe-Ser-Gly-Arg for M. fuscata and Asn-Glu-Glu-Val-Leu-Phe-Gly-Gly-Arg for E. patas. The sequence of the B peptide of M. fuscata differed from that of a close-related species, M. mulatta (rhesus monkey), at a single site, Leu (M.f.)----Pro (M.m.). A single replacement between the B peptides of E. patas and Cercocebus aethiops (green monkey), Val (E.p.)----Gly (C.a.), was detected. A phylogenic relationship of macaques, guenons, and baboons, named Cercopithecinae (Old World monkey), was deduced from the sequence data. A selective rather than random amino acid replacement was observed in the B peptides of these Old World monkeys, suggesting a restricted mutation of their fibrinopeptides during primate evolution.     

Amino acid sequence of polypeptide chain IIB of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus

The giant extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded trimer of chains IIA, IIB, and IIC. The complete amino acid sequence of chain IIB was determined. This chain has 148 amino acid residues and a molecular weight of 17,236 including a heme group. Of the residues in chain IIB, 74 (50%) and 34 (30%) were found to be identical with those in the corresponding positions in Tylorrhynchus chains IIC and I, respectively (Suzuki, T., Furukohri, T., and Gotoh, T. (1985) J. Biol. Chem. 260, 3145-3154). Marked differences were found between the chains of Tylorrhynchus and Lumbricus in the COOH-terminal regions. Significant differences were predicted between the monomeric chain I and the "trimeric" chains (IIB and IIC) in the hydropathy profiles and alpha-helical contents.     

A new type of blood group B active glycosphingolipid in rat bone marrow cells. Occurrence of the glycolipid in rat immunocytes and ascites hepatoma

A major glycosphingolipid in rat bone marrow cells was purified, and its structure was studied. The glycolipid was found to exhibit blood group B activity by the hemagglutination inhibition test. The structure was determined to be (formula; see text) by studies of nuclear magnetic resonance, sequential hydrolysis by exoglycosidases, linkage analysis of methylated sugars by gas chromatography-mass spectrometry, and immunological tests. The blood group B active glycolipid was detected not only in the bone marrow cells but also in spleen, thymus, and rat ascites hepatoma AH 7974F cells. Besides the glycolipid, gangliotriaosylceramide, gangliotetraosylceramide, and fucogangliotetraosylceramide were commonly detected in these cells. The similarity between the glycolipid species on the cell surfaces of the immunocytes and the tumor cells is discussed with the respect to an escape mechanism of the tumor cells from the immunosurveillance system.     

Combined effects of gamma rays, cis-diamminedichloroplatinum (CDDP) and systemic hyperthermia mastocytoma transplanted into muscle in the mouse

Comparison was made between the effects of local irradiation of gamma rays, s. c. injection of cis-diamminedichloroplatinum (II) (CDDP), systemic hyperthermia and their combinations on the i. m. transplanted murine mastocytoma. Increase of the mean survival time (M. S. T.) by a factor of 1.72 and of 1.68 was achieved by a single irradiation at 20 Gy, given on day 5 after transplantation, and by injections of CDDP at 2 mg/kg, given s. c. on days 5 and 12 after transplantation, respectively. Increase of M. S. T. by a factor of 1.10 which was achieved with systemic hyperthermia of 41.8 degrees C of the core body temperature for 5 min, given twice, on days 5 and 12 after transplantation, was not statistically significant. The most effective one among all possible combinations within the 3 modalities was that of radiation and CDDP. Increase of M. S. T. was by a factor of 4.01.     

In vitro absorption of inorganic phosphate and other electrolytes in the carp intestine

Net movements of water, Na, Cl, K, Ca, Mg and Pi were measured in the stripped and everted intestine of the carp. A high concentrative absorbtion of Pi was observed, whereas Na and Cl were absorbed almost isotonically. The rate of Pi absorption was greater in the middle part than in the anterior or posterior part of the intestine.