On the controversy over "fast" and "slow" helix-coil transition rates in polypeptides

The controversy over “fast” and “slow” helix-coil transition rates in polypeptides is discussed. The “slow” results are derived from the assumption that multiple NMR spectra of α-CH and NH groups arise from chemical exchange. In this paper it is shown that such spectra may be obtained without invoking chemical exchange. The multiplicity arises from the difference in helicity of amino acid residues near the ends of the chain by comparison with amino acid residues nearer the middle, and from a polydispersity in molecular weight. As a consequence of this analysis, support is given to the “fast” transition rates.     

Genetic demonstration of bidirectionality in the high affinity purine base transporter of mutant mouse S49 cells

A mutant cell line was selected from wild type S49 lymphoblasts that expressed a novel high affinity purine base transport system not found in parental cells or any other mammalian cell line (Aronow, B., Toll, D., Patrick, J., Hollingsworth, P., McCartan, K., and Ullman, B. (1986) Mol. Cell. Biol. 6, 2957-2962). In order to determine whether this nucleobase transport system was bidirectional, mutant cell lines possessing this high affinity base transport capability were derived from a nucleoside transport-deficient derivative of an adenylosuccinate synthetase-deficient S49 cell line. The resulting progeny excreted significantly greater amounts of purine into the cell culture medium than parental cells. This purine was identified as hypoxanthine. These results demonstrate genetically that the high affinity purine base transport system can mediate both the influx and efflux of hypoxanthine.     

Nutrient release rates from the sediments of Saginaw Bay,Lake Huron

Direct measurements of net production rates and pore water profiles of solutes in the fine-grained sediments of Saginaw Bay, imply corresponding steady-state fluxes to the overlying water of 1.1–1.3 (I), 450–1010 (NH₄ ⁺), 1250–2650 (Si(OH)₄), 3000–3400 (Ca²⁺), 440–1330 (Mg²⁺), 1.5–728 (Fe²⁺), and 179–281 (Mn²⁺) moles/m²/day and 11.0–11.8 (alkalinity) meq/m²/day at 17.5 °C. Silica production rates in sediments apparently follow first order kinetics with a rate coefficient of 0.09/day and a steady-state silica concentration of 1.2 mM at 23.5°C. The remaining solutes follow kinetics approximately independent of solute concentration over the range of concentrations observed. Measured solute production rates are consistent with observed solute profiles only if lateral diffusion gradients are maintained in the sediments by the burrowing and irrigation activity of benthic organisms such asChironomous, the dominant burrower in Saginaw Bay. Assuming that solute fluxes from Saginaw Bay are representative of all of the post-glacial sediments of Lake Huron, the iodine flux from sediments is comparable to the total fluvial input of iodine. The extrapolated silica fluxes from Lake Huron sediments balance the estimated biogenic silica flux to the sediments.     

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.     

Dipyridamole-insensitive nucleoside transport in mutant murine T lymphoma cells

From a mutagenized population of S49 murine T lymphoma cells, a mutant cell line, JPA4, was selected that expressed an altered nucleoside transport capability. JPA4 cells transported low concentrations of purine nucleosides and uridine more rapidly than the parental S49 cell line. The transport of these nucleosides by mutant cells was insensitive to inhibition by either dipyridamole (DPA) or 4-nitrobenzylthioinosine (NBMPR), two potent inhibitors of nucleoside transport in mammalian cells. Kinetic analyses revealed that the apparent Km values for the transport of uridine, adenosine, and inosine were 3-4-fold lower in JPA4 cells compared to wild type cells. In contrast, the transport of both thymidine and cytidine by JPA4 cells was similar to that of parental cells, and transport of these pyrimidine nucleosides remained sensitive to inhibition by both NBMPR and DPA. Furthermore, thymidine was a 10-12-fold weaker inhibitor of inosine transport in JPA4 cells than in wild type cells. Thus, JPA4 cells appeared to express two types of nucleoside transport activities; a novel (mutant) type that was insensitive to inhibition by DPA and NBMPR and transported purine nucleosides and uridine, and a parental type that retained sensitivity to inhibitors and transported cytidine and thymidine. The phenotype of the JPA4 cell line suggests that the sensitivity of mammalian nucleoside transporters to both NBMPR and DPA can be genetically uncoupled from its ability to transport certain nucleoside substrates and that the determinants on the nucleoside transporter that interact with each nucleoside are not necessarily identical.     

Biochemical phenotype of 5-fluorouracil-resistant murine T-lymphoblasts with genetically altered CTP synthetase activity

From wild type mouse lymphoma cells, a clone, (FURT-1A), was isolated by virtue of its resistance to 1 microM 5-fluorouracil and 10 microM thymidine. In comparative growth rate experiments, FURT-1A cells were also less sensitive than parental cells to the growth inhibitory effects of thymidine, deoxyguanosine, 5-fluorouridine, and arabinosylcytosine. The altered growth sensitivity of FURT-1A cells to cytotoxic nucleosides was directly related to their decreased ability to accumulate the corresponding triphosphate from exogenous nucleoside. FURT-1A cells contained elevated cytidylate nucleotide pools which prevented normal growth sensitivity and interfered with the salvage of nucleosides by inhibiting nucleoside kinase activities, by stimulating nucleotide dephosphorylating activities, and by overcoming certain allosteric inhibitions imposed on ribonucleotide reductase. Metabolic flux experiments with [3H]uridine in situ indicated that FURT-1A cells had a 2-fold enhanced rate of conversion of UTP to CTP. Kinetic analyses indicated that the CTP synthetase activity in extracts of FURT-1A cells was refractory to inhibition by CTP. The genetic loss of normal allosteric inhibition of the CTP synthetase activity in FURT-1A cells could account for the unusual phenotypic properties of these cells and conferred a high spontaneous mutator phenotype to cells possessing this mutation.     

Non-linearities in cortical simple cells and the possible detection of zero crossings

A theory of early visual information processing proposed by Marr and co-workers suggests that simple cortical cells may be involved in the detection of zero crossing in the retinal output. We have tested this theory by using pairs of adjacent edges (staircases stimuli) and recording from edge-specific simple cells in cat striate cortex. The zero crossing hypothesis gives rise for such stimuli to non-obvious predictions that were generally confirmed by the experiment.     

Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.

A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage.