Rapid group separations of nucleotides and related compounds on silica columns

Nucleosides, bases, and nucleotides can be separated from one another rapidly (10–15 min) on 1-ml silica cartridges. Samples adjusted to 4 mm ammonium borate, 90% acetonitrile are loaded onto 1-ml columns equilibrated with the same solvent. Bases do not absorb to the silica under these conditions. Nucleosides are eluted with 16 ml of 0.5 m acetic acid in 90% acetonitrile. Nucleotides are then eluted with water. The 1-ml silica columns have performed well with samples up to 10 ml in volume. We have found the procedure to be quantitative and the gels to have high capacity (61 μmol Cyd/ml silica). Acid extracts from a large number of cells (10⁸) have been processed on a single cartridge.     

Interaction of epidermal growth factor with micelles monitored by photochemically induced dynamic nuclear polarization-1H NMR spectroscopy

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.     

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Resistance of some common fungi to gamma irradiation

Ten species of fungi representing the genera Alternaria, Aspergillus, Caldosporium, Curvularia, Fusarium, and Penicillium were examined for their relative resistance to gamma irradiation from a 137Cs source. Inactivation doses for dematiaceous fungi in agar medium ranged from 0.6 to greater than 1.7 megarads, whereas those for moniliaceous fungi were less than 0.3 megarad. D10 values (the dose required to reduce the inoculum by 1 log) for Curvularia geniculata (greater than 0.29 megarad) exceeded those for control spores of Bacillus pumilus (0.15 megarad).     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone.

A novel cDNA was isolated from rat pituitary mRNA using the polymerase chain reaction to amplify sequences encoding G protein-coupled receptors. The human homolog of this cDNA was isolated and expressed in human kidney 293 cells, and membrane fractions from these cells were found to bind human GH-releasing hormone (GHRH) with high affinity and specificity. GHRH also stimulates intracellular cAMP production in these transfected cells. The encoded receptor protein contains seven potential membrane-spanning domains, a hallmark of G protein-coupled receptors, and is homologous to previously identified receptors for secretin and vasoactive intestinal peptide, ligands that are related to GHRH. The rat GHRH receptor mRNA is expressed predominantly, if not exclusively, in the anterior pituitary gland, the major target for GHRH action. These results define a mechanism for cellular signaling by GHRH and provide the opportunity to examine the role of the GHRH receptor in growth abnormalities that involve the GH axis.     

Photo-CIDNP nuclear magnetic resonance as a probe for conformational changes in epidermal growth factor

Photo-chemically induced dynamic nuclear polarization (CIDNP)-NMR spectroscopy at 360 MHz has been used to investigate pH-induced conformational transitions in mouse epidermal growth factor. At about pH 9, all five tyrosine residues and both tryptophan residues are, to various extents, solvent-exposed, while the His-22 residue is buried in the protein matrix. Tyr-13 is the least exposed of the tyrosine residues and also the most immobilized. As the pH is decreased to 5.9, the tryptophan residues gradually become less exposed, while the Tyr-13 residue becomes internalized in the protein. These data suggest that the C-terminus and part of the N-terminal structural domain are affected by a conformational transition in mouse epidermal growth factor occurring between pH 6 and 8 via breakage of the His-22 inter-residue linkage. Above pH 9, a decreased photo-CIDNP effect is evident for both tryptophans and for Tyr-10 and Try-13; this information suggests that a second conformational change takes place at basic pH, which may simply be incipient denaturation.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.