Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS

A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a approximately 200-500-microm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-microm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-microl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-microl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.     

An intercomparison of sampling techniques for measurements of radiocaesium in upland pasture and soil

An intercomparison of sampling procedures used by five laboratories for the determination of radiocaesium in vegetation and peaty soil was carried out at two locations in Cumbria. The multiple sampling has given information on the homogeneity of the parameters studied at each location. The parameters comprise soil bulk densities, total deposition of¹³⁷Cs, deposition of¹³⁷Cs in three soil layers, biomass densities, concentrations of¹³⁷Cs in pasture, and activity ratios (¹³⁴Cs/¹³⁷Cs) in soil and vegetation. The determination of total deposition of¹³⁷Cs gave no indication of differences between the laboratories. The results from the soil profiles do indicate significant differences between laboratories. One laboratory using a coring technique observed difficulties during sampling due to compression of the soil. The coring technique should thus be avoided or applied with extreme care for the sampling of depth profiles in peaty soil. The results from the sampling of pasture show no indication of differences between the laboratories. For the parameters studied the observed variabilities across soil depths and locations range from 10% to 81% in terms of relative standard deviations. A comparison across all results at the two locations indicate a 50% higher field variability at one of the sites relative to the other.     

THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract. The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235–302 nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.     

Synthesis and burst strength of water‐swollen immunoisolatory tubules

A series of amphiphilic hydrogel tubules have been prepared by copolymerizing/crosslinking hydrophilic poly(dimethylacrylamide) segments with hydrophobic di‐, tri‐, and octamethacrylate‐telechelic polyisobutylene crosslinkers, and their elastic modulus and burst strength in the water‐swollen state were investigated. Because the burst characteristics of hydrogels have not yet been quantitatively investigated, equipment was designed and built to generate fundamental insight into the burst properties of thin‐walled (200–250 μm) narrow lumen (2–3 mm i.d.) water‐swollen tubules. The theory developed to describe quantitatively the inflation behavior of thin‐walled rubber tubules was adapted to treat our experimental observations. Changes in the burst strength, elastic modulus, and expansion during the inflation of hydrogel tubules were interpreted in terms of the molecular weight of the hydrophilic segments between crosslinking sites (M_{c,hydrophilic}), which in turn was calculated according to the rubber elasticity theory. According to these investigations, the burst strength of our water‐swollen amphiphilic tubules is in the 0.2–0.5 MPa range, which is sufficient for implantation and immunoisolatory applications. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 2075–2084, 2002     

Isolation of ammonium-N as 1-sulfonato-iso-indole for measurement of delta15N

The conversion of ammonium (NH(4) (+)) to 1-sulfonato-iso-indole has been examined as a method for natural abundance measurement of delta(15)N of NH(4) (+). The reaction is complete within 2 h and is based on the derivatisation of NH(4) (+) by o-phthaldialdehyde and sodium sulfite at a high pH, 11.2. The product is readily concentrated from dilute solutions by reverse-phase solid-phase extraction (SPE). The method is compound-specific despite partial derivatisation of potentially interfering amino acids, as their derivatives are not extracted by SPE. delta(15)N values of NH(4) (+) in KCL soil extracts can be measured within 48 h by automated continuous-flow IRMS with a precision of 0.23 per thousand (1 SD). Parallel measurements of NH(4) (+) standards of known delta(15)N are made to allow correction for the isotopic dilution by non-sample NH(4) (+). The practicality of this method is demonstrated by measuring the changes in NH(4) (+) concentration and delta(15)N following the addition of urea as a nitrogen source to inorganic N-depleted soil.     

Reaction of carbonyl fluoride with fluorine in the presence of various fluorides as catalysts

The fluorides KF, RbF and CsF have been known to serve as catalysts for the reaction CF₂O + F₂→ CF₃OF. The list of catalysts for this process has now been enlarged to include NaF, MgF₂, CaF₂, SrF₂, BaF₂ and LaF₃. Lithium fluoride and thorium fluoride also give CF₃OF but are less active. Perhaps the substances CsF·HF, KAgF₄ and NiF₂ should be included in this list. Silver fluoride, usually as a mixture of AgF₂ with AgF, has been known to catalyze the reaction of CF₂O with F₂ to give both CF₃OF and CF₃OOCF₃. The proportion of the latter in the mixture of products increases with decreasing temperature. At 25°, the reaction is slow and the yield of CF₃OOCF₃ is very high. It has now been shown that TIF₃ behaves like silver fluoride. It has also been shown that many other fluorides of metals give higher yields of CF₃OOCF₃ than of CF₃OF but require higher temperatures than AgF₂ (100-ca. 150°) to be effective. Various possible mechanisms for these catalytic processes are discussed.     

Dramatic 5'-residue effect on conformer distribution of short oligonucleotide retro models of the cisplatin-DNA cross-link: implications for the Lippard and cross-link distorted base pair steps present in cisplatin-DNA duplex adducts

The cisplatin anticancer drug preferentially attacks the GG sequence of DNA duplexes. Virtually all DNAs containing the key G*G* lesion (G* = N7 platinated G) have large distortions in the cross-link (G*G*) base pair (bp) step and also in the adjacent Lippard (XG*) bp step, making the adducts very different from B-form DNA in the XG*G* region. The XG*G* strand in duplexes also differs in several ways from single-strand (ss) models with G*G* and XG*G* sequences. In the duplex, the X residue has an N sugar, the 5'-G* and 3'-G* bases have slight "R" canting (3'-G* H8 atom toward the 5'-G* base), and there is no or weak H-bonding by the NH3 ligands. In most XG*G* ss models, X has an S sugar, the 5'-G* base normally cants strongly toward the 3'-G* base (L canting), and the NH3 forms an H-bond. Well-defined ss models exist in the solid state, but dynamic motion obscures the properties of the ss models in solution. In this work, we employ retro models (better defined, less dynamic ss models) to understand the differences between duplex and ss models. The retro models in this study lack carrier ligand NH's, thus eliminating H-bonding. To correlate previous ss solid-state models with our solution work, we constructed hybrid molecules by overlaying parts of known structures. The combined model and experimental information indicates that the X N-pucker is not favorable in L-canted ss models, that X residue steric effects (not H-bonding) favor L canting in ss models, that X N-pucker is needed for favorable WC hydrogen bonding and stacking interactions in duplexes, and that X N-pucker minimizes X base clashes with bases in the complementary strand in duplexes. The R canting minimizing clashes between the X and G* residues of the Lippard bp step (independent of X pucker) and the repositioning of the X residue base caused by the change from S-pucker to N-pucker together lead to the unusual features of the Lippard bp step in the duplex.     

SV40 INDUCTION FROM A MAMMALIAN CELL LINE BY ULTRAVIOLET RADIATION and THE PHOTOSENSITIZERS 8-METHOXYPSORALEN and ANGELICIN

Abstract SV40-transformed weanling Syrian hamster kidney cells were exposed to long wavelength ultraviolet radiation in the presence of 8-methoxypsoralen or angelicin. The number of viruses induced as a result of this treatment was quantitated on CV-1P cells. The results indicate that angelicin, which is generally believed to form only monoadducts, is a potent viral inducing agent even when compared with 8-methoxypsoralen (8-MOP), which forms both monoadducts and crosslinks. At low radiation exposures 8-MOP is more effective than angelicin; as the radiation exposure is increased angelicin is capable of inducing the same peak level of virus expression as was 8-MOP. It appears that the induction of SV40 is a phenomenon possibly related to the repair of monoadducts.     

Stereoselective enolizations mediated by magnesium and calcium bisamides: contrasting aggregation behavior in solution and in the solid state

The reactions of magnesium and calcium bis(hexamethyldisilazide) with propiophenone have been studied with a view to determine the utility of these bases in the stereoselective enolization of ketones and to uncover the nature of the metal enolate intermediates produced. Both base systems are highly Z-selective when the reactions are conducted in the presence of polar solvents. However, in situ monitoring of the magnesium system in arene solution revealed a preference for E-enolate formation, which was confirmed by silyl enol ether trapping studies. Solution NMR studies of the magnesium system in toluene-d8 show the presence of a monomer-dimer equilibrium for the intermediate amidomagnesium enolates. This assignment is supported by the characterization of a disolvated amidomagnesium enolate dimer by crystallographic analysis. Comparative studies of the calcium system show distinctly different behavior. This is exemplified by the characterization of a novel solvent-separated ion pair complex and a monomeric amidocalcium enolate in the solid state. Solution NMR studies of the calcium system in pyridine-d5 reveal the co-existence of the heteroleptic amidocalcium enolate, the bisamide, the bisenolate and the ion pair complex.     

CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5-6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero- and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N-methylated porphyrins show clearance times in the vicinity of 15-22 h. Monoaspartyl chlorin e6 shows the longest clearance time of all porphyrin derivatives measured (30.3 h).