The Amino Acid Sequence of the Tryptophan-Containing Subunit (α-Subunit) of Bovine Brain S-100 Protein

Abstract: The tryptophan-containing subunit (α-subunit) of bovine brain S-100 protein was purified from a S -aminoethyl derivative of S-100a protein, and its amino acid sequence was determined. The α-subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another "tryptophan-free" subunit (β-subunit) found in both S-100a and S-100b protein, and has a calcium binding site characteristic of the "E-F hand" proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C-terminal end of the α-helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure-function studies of S-100a protein.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Angeloylcumambrin-B,an antimicrobial sesquiterpene lactone from Chrysanthemum ornatum var. Spontaneum

Angeloylcumambrin-B, a new antimicrobial guaianolide sesquiterpene lactone, was isolated from Chrysanthemum ornatum and the structure was determined by a combination of chemical and physical methods.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. Role of cardiolipin in fluidity of mitochondrial membranes

During temperature acclimation of Tetrahymena pyriformis, the changes in fluidity and composition of total lipids from three membrane fractions, mitochondria, pellicles and microsomes were studied by a spin-label technique using a stearate probe and thin-layer and gas-liquid chromatography. The increase of fluidity observed in microsomal and pellicular lipids following the temperature shift from 39 to 15 degrees C corresponds with the increase of the ratio of total unsaturated to saturated fatty acid content. However, despite the increase of this ratio, the fluidity of mitochondrial lipids was found to be constant up to 10 h after the temperature shift. The fluidity of total lipids of mitochondria isolated from Tetrahymena cells grown at 39 degrees C was not changed by removal of cardiolipin, whereas cardiolipin-depleted lipids of mitochondria from 15 degrees C-acclimated cells showed a decrease in fluidity. The re-addition of cardiolipin to the mitochondrial lipids depleted of cardiolipin restored the fluidity to the initial level, thereby confirming the rigidifying effect of cardiolipin in cold-acclimated cells. These results suggest that cardiolipin may be implicated in maintaining consistent fluidity of mitochondrial membranes against change in thermal environment.     

Rheological studies on calcium-sensitive gelation of actin filaments by actinogelin

Actinogelin, a regulatory protein of cell motility, enhanced gelation of actin filaments in the absence of calcium ions, only on standing still or with very low velocity gradients ( less than 0.1 s-1). The Ca2+-sensitive action of actinogelin on action filaments was dependent on a weak external force. In the presence of a micromolar level of Ca2+, actinogelin did not affect the network formation of actin filaments at all.     

Cell-cycle-specific inhibition by chloramphenicol of septum fromation and cell division in synchronized cells of Bacillus subtilis.

The relationship between protein synthesis and processes of cell division was studied by using synchronized cells of Bacillus subtilis 168. The addition of chloramphenicol at the beginning of synchronous growth prevented septum formation and cell division, suggesting the requirement of protein synthesis for the processes of cell division. Experiments in which the drug was added to the cells at different cell ages showed that the protein synthesis required for the initiation of septum formation was completed at about 15 min and that the protein synthesis required for cell division was completed at about 45 min. By interpreting the result from the concept of the transition point for protein synthesis, it was suggested that the processes of cell division in B. subtilis require at least two kinds of protein molecules which are synthesized at distinct stages in the cell cycle. This was supported by the result of an experiment in which starvation and the readdition of a required amino acid to exponentially growing cells induced two steps of synchronous cell division. Further, the two transition points are in agreement with the estimations obtained by residual division after the inhibition of protein synthesis in asynchronous cells. The relationship of the timing between the completion of chromosome replication and the two transition points was also studied.     

Hemoglobins of the tadpole of the bullfrog, Rana catesbeiana. Amino acid sequence of the alpha chain of a major component

The complete amino acid sequence has been determined for the alpha chain of component III of the hemoglobin of the tadpole of the bullfrog, Rana catesbeiana. The chain comprises 141 residues of which 80 (57%) are identical to those in the corresponding positions of the human chain. Almost the same extent of similarity exists in the comparison with the sequenced part of the alpha chain of the adult bullfrog. The major features of this chain are: 1) each residue which is common to all other alpha chains of known sequence is also found in this alpha chain; 2) an acetylated NH2 terminus prevents formation of one of the salt bridges found in human hemoglobin which is responsible for part of the alkaline Bohr effect in mammalian hemoglobins; and 3) a prolyl residue at alpha 99 (G6) must distort the G helix.     

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

The oxidative cleavage of folates. A critical study

Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C⁹N¹⁰ bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N⁵-formyl-tetrahydrofolic acid are cleaved at the C⁹N¹⁰ bond to produce p-aminobenzoylglutamic acid. N⁵, N¹⁰-methenyl-tetrahydrofolic acid, N⁵,N¹⁰-methylene-tetrahydrofolic acid, and N¹⁰-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N¹⁰-formyl-folic acid which is completely stable to the oxidative treatment employed. N⁵-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N⁵-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-³H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-¹⁴C]folic acid to label the folates in vivo is presented.