High-performance liquid chromatographic analysis of neutral glycosphingolipids as their per-O-benzoyl derivatives

Previous reports from this laboratory (1–4) described the perbenzoylation of neutral glycosphingolipids (GSL)¹ with benzoyl chloride in pyridine and analysis of the perbenzoylated derivatives by high performance liquid chromatography (hplc). A disadvantage of this procedure is that N-benzoylation occurs as well as the desired O-benzoylation. This does not permit recovery of the parent GSL after mild alkaline hydrolysis due to formation of a mixture of N-acylated and N-benzoylated GSLs(1). It has also been demonstrated that the benzoylation with benzoic anhydride in pyridine does not lead to the formation of N-benzoylated products. However, the anhydride reaction is sluggish and the benzoyl chloride method has been the preferred procedure.Gupta et al. (5) used N,N-dimethyl-4 amino pyridine (DMAP) as a catalyst in the acylation of phospholipids by the anhydrides of fatty acids. F. B. Jungalwala (private communication) has shown that this catalyst greatly accelerates the reaction of benzoic anhydride with sulfatides.In this communication we report the preparation and hplc analysis of per-O-benzoyl derivatives of GSLs by reaction with benzoic acid anhydride in the presence of DMAP as a catalyst. Reaction with these reagents avoids amide acylation, forms single products with satisfactory chromatographic properties and parent GSLs can be regenerated by mild alkaline hydrolysis.     

Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts.

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.     

The amino acid sequence of chick skin collagen α1-CB7: The presence of a previously unrecognized triplet

Because alignment of the amino acid sequences of chick skin collagen α2-CB3 (1) with the relevant portion of chick skin collagen α1-CB7 (2) suggested that a Gly-X-Y triplet may have been missed in the latter, the peptide TM-2, produced by tryptic digestion of maleylated α1-CB7, was reinvestigated. Cleavage by trypsin at the unblocked lysine at position 18, and isolation of the resulting COOH-terminal peptide, showed this to be a 15-residue peptide containing a previously unrecognized Gly-Pro-Hyp triplet. Sequencing of the peptide showed this to occupy positions 4 through 6, or 56 through 58 of α1-CB7. The latter thus has 271 instead of 268 residues, and the α1[I] chain 1055 instead of 1052.     

Phase response versus positive and negative division delay in animal cells

The time of median cell division in V79 Chinese hamster cells following high serum pulses was determined for two synchronous cell generations following mitotic selection. Differences in cell cycle time for each pair of pulse and control cultures were computed and plotted as a function of time of serum pulse. This phase response curve for hamster cells with an 8.5 h cell cycle shows a characteristic biphasic pattern. Beginning 0.5 h after mitotic selection, pulses with serum produce delays in the midpoint of the subsequent mitotic waves. Delay is maximum at 1.5 h. Delays give way abruptly to advances at 2.5 h and the amount of advance then decreases as pulses are given between 3 and 5 h into the cycle. At 5 h decreasing advances become delays, with increasing delays due to serum pulses occurring between 5 and 6 h. Delays again give way abruptly to advances at 6 h and again the amount of advance decreases through the late portion of the cycle. Pulses very late in the cycle appear to generate phase delays. This biphasic response to serum is interpreted as an expression of an underlying time-keeping oscillator whose period is nominally of 4 h duration.     

Signal emission and signal propagation during early aggregation in Dictyostelium discoideum.

Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum are of 2 kinds, concentric waves produced by cells that emit cyclic AMP signals spontaneously, and spirals generated by excitations relayed continuously around loops of excitable cells. The period of a spiral wave is the time taken for the excitation to make one complete circuit of the pacemaker loop. We have compared signal emission from the 2 types of source in time-lapse films made at a variety of temperatures. Our results show that spiral waves have a characteristic period length throughout most if not all of the early phase of aggregation, and that the period of concentric waves is generally longer and more variable. Temperature has a pronounced effect on period length and a lesser effect on propagation velocity. We find that each individual wave is propagated at constant velocity over distances of 1-2 cm but that the velocity of successive waves declines. This decline probably reflects some cumulative effect of the chemotactic excitations on the excitable properties of the aggregating cells.     

Drug-induced handwriting changes: an empirical review.

Since handwriting is a highly complex, coordinated motor activity, handwrits of pharmacological agents. Its potential has been most evident in research involving therapeutic administrations of anti-psychotic an anti-Parkinsonian drugs, from which consistent and systematic handwriting changes have been observed. This relationship has been found to be particularly significant among the anti-psychotics, since the onset of these graphomotor alterations appear to mark the optmal dose of the drug. Consistent and systematic handwriting changes have not been as evident in inivestigations of drugs used in a nontherapeutic atmosphere. Psychiatric assessments of subjects in ths type of research provided data which indicated that psychological stability may be a factor influencing the susceptibility of one's handwriting to drug induced changes.