Composition and Metabolism of Gangliosides in Rat Peripheral Nervous System During Development

Abstract: Ganglioside composition of rat trigeminal nerve was studied during development in order to understand the changes that occur as a result of cellular differentiation in the nerve. The ganglioside composition of the trigeminal nerve was entirely different from that of brain. The major gangliosides in adult trigeminal nerve were GM₃, GD₃, and LM₁ (sialosyl-lactoneotetraosylceramide or sialosylparagloboside). The structure of LM₁ and other gangliosides was established by enzymatic degradation and by analysis of the products of acid hydrolysis. At 2 days after birth, when the Schwann cells were immature, GM₃ and GD₃ were the major gangliosides in the nerve, 50 and 18 mol %, respectively. As the nerve developed and Schwann cells proliferated and myelinated the axons, the mol % of GM₃ and GD₃ reduced and that of LM₁ steadily increased. Polysialogangliosides did not change drastically with nerve development. The rate of deposition of LM₁ in the nerve with age was very similar to that of myelin marker lipids, cerebrosides, and sulfatides; thus, deposition appears to be localized mainly in the rat nerve myelin. LM₁ also had long-chain fatty acids 22:0 and 24:0, which are not usually found in CNS gangliosides. The ganglioside pattern of the rat trigeminal nerve was very similar to that of rat sciatic nerve, but was different from that of rabbit and chicken sciatic nerve. The activity of the two key enzymes involved in the metabolism of GM₃, viz., CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase and UDP-N-acetylgalactosamine:GM₃-N-acetylgalactosaminyltransferase, was also studied during development of the nerve and brain. The developmental profiles of both enzymes were consistent with the amounts of GM₃ present in the nerve.     

Prediction of beta-turns.

An automated computer prediction of the chain reversal regions of globular proteins is described herein using bend frequencies and beta-turn conformational parameters (Pt) determined from 408 beta-turns in 29 proteins calculated from x-ray atomic coordinates. The probability of bend occurrence at residue i is pt = fi X fi+1 X fi+2 X fi+3 with the average bend probability less than Pt greater than = 0.55 X 10(-4). Tetrapeptides with pt greater than 0.75 X 10(-4) ( approximately to 1.5 X less than pt greater than) as well as less than Pt greater than 1.00 and less than Pa greater than less than less than Pt greater than greater than less than P beta greater than are selected by the computer as probable bends. Adjacent probable bends (i.e., 11-14, 12-15, 13-16) are compared pairwise by the computer, and the tetrapeptide with the higher pt value is predicted as a beta-turn. The percentage of bend and nonbend residues predicted correctly for 29 proteins by this computer algorithm is %t+nt = 70%, whereas 78% of the beta-turns were localized correctly within +/- 2 residues. The average beta-turn content in the 29 proteins is 32%, with helical proteins having fewer bends (17%) than beta-sheet proteins (41%). Three proteins having iron-sulfur clusters were found with the highest percentages of beta-turns: Chromatium high potential iron protein (65%), ferredoxin (57%), and rubredoxin (65%). Finally, the bend frequencies at all 12 positions from 457 beta-turns in 29 proteins (Chou and Fasman, 1977) were used to test the effectiveness of predicting bends using 2, 4, 8, and 12 residues as well as different cut-off pt values. The computer analysis showed that 1.25 less than pt greater than to be the best cut-off yielding 70% accuracy in %t+nt for 4 residues and %t+nt = 73% for 12 residues in predicting the bend and nonbend regions of proteins.     

Conservation of chain reversal regions in proteins.

Using the bend frequencies based on 29 proteins in the previous paper (Chou and Fasman, 1979), beta-turn probability profiles were calculated for the C-peptides of 10 mammalian proinsulins, for 7 proteinase inhibitors, and for 12 species of pancreatic ribonucleases. Beta-turn correlation coefficient matrix tables were also computed to obtain the statistical mean between 45 pairs of proinsulin C-peptides, less than Ct greater than = 0.57 +/- 0.31; 21 pairs of proteinase inhibitors, less than Ct greater than = 0.73 +/- 0.13; and 66 pairs of ribonucleases, less than Ct greater than = 0.83 +/- 0.08. Despite relatively low sequence conservation in these three sets of proteins, beta-turns were predicted to be highly conserved: 33% sequence vs. 78% bend for the proinsulins, 20% sequence vs. 85% bend for the proteinase inhibitors, and 65% sequence vs. 92% bend for the ribonucleases. These results suggest that chain reversal regions play an essential role in keeping the active structural domains in hormones and enzymes intact for their specific biological function.     

Simian virus 40 mutants carrying two temperature-sensitive mutations.

Five temperature-sensitive mutants of simian virus 40 containing two temperature-sensitive mutations were isolated. The double mutant of the A and D complementation groups, like the D mutants, failed to complement by conventional complementation analysis and did not induce host DNA synthesis at 40 degrees C. However, under conditions that suppressed the D defect, the A:D double mutant expressed only the A defect. Thus, viral DNA replication dropped rapidly after this mutant was shifted from permissive to restrictive temperatures. The A:D double mutant failed to transfrom at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. Double mutants of A:B, A:C, and A:BC complementation groups, like their A parent, were defective in viral DNA replication, in the induction of host DNA synthesis and in the transformation of secondary Chinese hamster lung cells at the nonpermissive temperature.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.     

The conformation of glucagon: predictions and consequences.

It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.     

Simian virus 40 functions required for the establishment and maintenance of malignant transformation.

Members of the five classes of temperature-sensitive simian virus 40 mutants were tested for their ability to transform Chinese hamster lung cells. Two criteria for transformation were used: the ability to form clones in medium with low serum concentrations and the ability to overgrow a monolayer. Only A mutants failed to transform at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. However, both A and D mutants failed to transform at the restrictive temperature when confluent monolayers and depleted medium were used. When transformed clones were selected, purified by recloning, and examined at both temperatures, only cell lines induced by A mutants lost the transformed phenotype at the higher temperature. Thus, A function is required for maintenance of the transformed phenotype in Chinese hamster lung cells. A function is known to be required for the initiation of viral DNA synthesis in permissive cells. Therefore, transformation may be a consequence of the introduction into a cell of the capacity for aberrant initiation of DNA replication.     

Products of complementation between temperature-sensitive mutants of simian virus 40.

Temperature-sensitive mutants of the D complementation group of simian virus 40 exhibit delayed complementation. Analysis of the thermal stability, kinetic profiles in temperature shift experiments, and progeny of complementation have led to the hypothesis that delayed complementation is not true complementation, but the result of a very low level of leakiness, followed by phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants at the restrictive temperature.     

Frequent Unanticipated Alleles of lethal giant larvae in Drosophila Second Chromosome Stocks

Forty years ago, a high frequency of lethal giant larvae (lgl) alleles in wild populations of Drosophila melanogaster was reported. This locus has been intensively studied for its roles in epithelial polarity, asymmetric neural divisions, and restriction of tissue proliferation. Here, we identify a high frequency of lgl alleles in the Bloomington second chromosome deficiency kit and the University of California at Los Angeles Bruinfly FRT40A-lethal P collection. These unrecognized aberrations confound the use of these workhorse collections for phenotypic screening or genetic mapping. In addition, we determined that independent alleles of insensitive, reported to affect asymmetric cell divisions during sensory organ development, carry lgl deletions that are responsible for the observed phenotypes. Taken together, these results encourage the routine testing of second chromosome stocks for second-site alleles of lgl.     

Absence of radiofrequency heating from auditory implants during magnetic resonance imaging

The possibility of tissue heating due to an auditory brainstem implant (ABI) or a modified cochlear implant (CI) during magnetic resonance imaging (MRI) of the head was tested on a full-sized human phantom using a realistic phantom head consisting of simulated skull, brain, and muscle. Dielectric properties of the brain, muscle, and bone materials were similar to those of human tissues at 64 MHz. The body consisted of homogeneous phantom muscle enclosed in a human-shaped fiberglass shell. Thermographic and fiber-optic temperature measurements were conducted to reveal any heating. Thermograms of sagittal, frontal, and horizontal planes of the head with the ABI and CI electrodes were taken immediately before and after a 26 min MRI scan. The MRI sequence was set at 94 excitations and 25 ms echo time to induce maximum radiofrequency heating, as suggested by the General Electric Company. The difference of these two thermograms gives the heating results. In two uncut phantom heads, Teflon tubes were placed along the implanted ABI and CI, and temperature data were recorded via fiber-optic probes before, during, and after the MRI. Results showed no observable heating associated with the ABI and the modified CI during worst-case MRI of the head. © 1995 Wiley-Liss, Inc.