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Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Large Alterations in Ganglioside and Neutral Glycosphingolipid Patterns in Brains from Cases with Infantile Neuronal Ceroid Lipofuscinosis/Polyunsaturated Fatty Acid Lipidosis

Lipid composition was studied on cerebral tissue from nine children who had died of a progressive encephalopathy called the infantile form of neuronal ceroid lipofuscinosis (INCL) or polyunsaturated fatty acid lipidosis (PFAL). In the terminal stage of the disease, the concentrations of all lipid classes were found to be significantly reduced in the cerebral and cerebellar cortex and white matter. The concentration of gangliosides of the cerebral cortex was 15% and that of cerebrosides (galactosylceramide) in white matter 0.2-5% of the normal values for the children's ages. The reduction of gangliosides mainly affected those of the gangliotetraose series, particularly GD1a. The fatty acids of the linolenic acid series were strongly reduced in ethanolamine and serine phosphoglycerides. A very large increase up to 100-fold of oligoglycosphingolipids of the globo series and two fucose-containing lipids of the neolacto series was found in the forebrain of the three advanced cases examined. The brain tissue also contained very high concentrations of mono-, di-, and trisialogangliosides of the lacto and neolacto series, gangliosides with type 1 chain dominating. The structures of the gangliosides were tentatively identified by gas chromatography-mass spectrometry and monoclonal antibodies with carefully determined epitope specificity. The gangliosides and neutral glycosphingolipids had very similar fatty acid composition, consisting of about 40% stearic acid and 40% C24-acids.     

Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli

The bacterial membrane serves both as a cell organelle and as a barrier for segregating the metabolically active cytoplasm from the extracellular milieu. Thus we can use plasmid vectors designed to produce a hybrid protein containing an efficient signal peptide coupled to the amino terminus of the cloned heterologous protein (secretion cloning vectors) for the production of proteins which are insoluble, proteolytically sensitive, or bacteriocidal when produced in the cytoplasm of Escherichia coli. We demonstrate that human granulocyte-macrophage colony stimulating factor can be isolated as an active species only after transport into the bacterial periplasm. Production of the protein in the bacterial cytoplasm is bacteriocidal. We also demonstrate that biologically active human interleukin 4 appears only after transport of the protein into the bacterial growth medium. The protein forms membrane-associated aggregates in the cytoplasm, and demonstrates an active but nonnative conformation when expressed in the periplasm. This may correlate with the affinity of the interleukin 4 molecule for negatively charged macromolecules, including bacterial membrane components and bacterial lipopolysaccharides, which may alter the folding pathway inside the cell.     

Structural studies on cyanobacterial photosystem I

The cyanobacterial photosystem, I complex from Synechococcus sp. PCC6301 contains polypeptides of apparent M_r of 70,000, 18,000, 17,700, 16,000 and 10,000. Procedures were developed for the purification of the M_r 17,700 and 10,000 polypeptides. Amino acid analyses showed the absence of cystine and cysteine from these polypeptides. Amino-terminal sequences of 98 residues for the M_r 17,700 polypeptide and of 42 residues for the M_r 10,000 polypeptide were determined. Studies of pigment distribution within the photosystem I complex indicated that the binding of chlorophyll a and -carotene is in part dependent on the presence of these polypeptides.Abbreviations PSI photosystem I - P700 reaction center of PSI - SDS sodium dodecylsulfate - TBS tris-buffered saline - TTBS TBS containing Tween-20     

Catalytic mechanisms and regulation of lignin peroxidase.

Lignin peroxidase (LiP) is a fungal haemoprotein similar to the lignin-synthesizing plant peroxidases, but it has a higher oxidation potential and oxidizes dimethoxylated aromatic compounds to radical cations. It catalyses the degradation of lignin models but in vitro the outcome is net lignin polymerization. LiP oxidizes veratryl alcohol to radical cations which are proposed to act by charge transfer to mediate in the oxidation of lignin. Phenolic compounds are, however, preferentially oxidized, but transiently inactivate the enzyme. Analysis of the catalytic cycle of LiP shows that in the presence of veratryl alcohol the steady-state turnover intermediate is Compound II. We propose that veratryl alcohol is oxidized by the enzyme intermediate Compound I to a radical cation which now participates in charge-transfer reactions with either veratryl alcohol or another reductant, when present. Reduction of Compound II to native state may involve a radical product of veratryl alcohol or radical product of charge transfer. Phenoxy radicals, by contrast, cannot engage in charge-transfer reactions and reaction of Compound II with H2O2 ensues to form the peroxidatically inactive intermediate, Compound III. Regulation of LiP activity by phenolic compounds suggests feedback control, since many of the products of lignin degradation are phenolic. Such control would lower the concentration of phenolics relative to oxygen and favour degradative ring-opening reactions.     

Azotobacter vinelandii ferredoxin I: cloning, sequencing, and mutant analysis

The structure of Azotobacter vinelandii ferredoxin I (AvFdI) has been extensively characterized by a variety of techniques. Although its physiological function is unknown, it has long been implicated as being involved in electron donation to nitrogenase. Here we report that the AvFdI gene (fdxA) has been cloned from an EcoRI digest lambda library using a synthetic oligonucleotide probe and that its sequence has been determined. The amino acid sequence deduced from the DNA sequence is identical to the previously published protein sequence. Analysis of the promoter region indicates that AvFdI is not a nif specific gene product. A mutant of A. vinelandii has been constructed which is identical to the wild-type, at the DNA level, except that the fdxA gene has been interrupted by insertion of a kanamycin cartridge. This mutant, called LM100, does not synthesize AvFdI but does synthesize the Fe and MoFe proteins of nitrogenase and grows at wild-type rates under N2-fixing conditions. This demonstrates that AvFdI is not required for N2 fixation by A. vinelandii. There is a small acidic protein, which is present in wild-type A. vinelandii, whose level is dramatically increased in LM100. The nature of this protein is under further investigation.     

Nitrogen in shoot litter,root litter and root exudates from nitrogen-fixingAlnus incana

Summary A pot experiment withAlnus incana (L.) Moench growing in sand was set up to compare the amounts of nitrogen released from plants shoot litter with that released below ground as root litter and/or root exudation. No nitrogen fixation by free-living microorganisms was found in the sand and the increased nitrogen content of the plant + soil system was therefore due to nitrogen fixation byFrankia in the alder root-nodules. Most of the nitrogen released from the plants was in the nitrogen-rich leaf and other shoot litter. Only small amounts of nitrogen were found in the drainage water from the pots and were recorded as increased nitrogen content of the sand.