Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F2-isoprostanes in human urine

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF2α—F2-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F2-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F2-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F2-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F2-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF2α-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F2-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F2-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF2α-VI in hypercholesterolemia and of 8,12-iso-iPF2α-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.

Drosophila host defense: Differential induction of antimicrobial peptide genes after infection by various classes of microorganisms

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.

Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants

Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies). Analysis in Saccharomyces cerevisiae of a broad range of human familial amyotrophic lateral sclerosis–linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals in vivo. Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants. The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes. The mutant enzymes also are shown to acquire the catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

Identification of Discrete Classes of Endosome-derived Small Vesicles as a Major Cellular Pool for Recycling Membrane Proteins

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5′-[γ-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.

Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences

The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions. The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements. Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends. With minor exceptions the relationships range from 80% to 100% match within blocks. The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered “single-copy” (or better “two-copy”) sequences. All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed. It appears there has been an extensive history of sequence exchange or copying between terminal regions. The subtelomeric sequences fall into two classes. Seventeen of the chromosome ends terminate with the Y′ repeat, while 15 end with the 800-nt “X2” repeats just adjacent to the telomerase simple repeats. The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent.

Local translation of classes of mRNAs that are targeted to neuronal dendrites

The functioning of the neuronal dendrite results from a variety of biological processes including mRNA transport to and protein translation in the dendrite. The complexity of the mRNA population in dendrites suggests that specific biological processes are modulated through the regulation of dendritic biology. There are various classes of mRNAs in dendrites whose translation modulates the ability of the dendrite to receive and integrate presynaptic information. Among these mRNAs are those encoding selective transcription factors that function in the neuronal soma and ionotropic glutamate receptors that function on the neuronal membrane. Conclusive evidence that these mRNAs can be translated is reviewed, and identification of the endogenous sites of translation in living dendrites is presented. These data, as well as those described in the other articles resulting from this colloquium, highlight the complexity of dendritic molecular biology and the exquisitely selective and sensitive modulatory role played by the dendrite in facilitating intracellular and intercellular communication.

Different sources of reduced carbon contribute to form three classes of terpenoid emitted by Quercus ilex L. leaves.

Quercus ilex L. leaves emit terpenes but do not have specialized structures for terpene storage. We exploited this unique feature to investigate terpene biosynthesis in intact leaves of Q. ilex. Light induction allowed us to distinguish three classes of terpenes: (i) a rapidly induced class including alpha-pinene; (ii) a more slowly induced class, including cis-beta-ocimene; and (iii) the most slowly induced class, including 3-methyl-3-buten-1-ol. Using 13C, we found that alpha-pinene and cis-beta-ocimene were labeled quickly and almost completely while there was a delay before label appeared in linalool and 3-methyl-3-buten-1-ol. The acetyl group of 3-methyl-3-buten-1-yl acetate was labeled quickly but label was limited to 20% of the moiety. It is suggested that the ocimene class of monoterpenes is made from one or more terpenes of the alpha-pinene class and that both classes are made entirely from reduced carbon pools inside the chloroplasts. Linalool and 3-methyl-3-buten-1-ol are made from a different pool of reduced carbon, possibly in nonphotosynthetic plastids. The acetyl group of the 3-methyl-3-buten-1-yl acetate is derived mostly from carbon that does not participate in photosynthetic reactions. Low humidity and prolonged exposure to light favored ocimenes emission and induced linalool emission. This may indicate conversion between terpene classes.

Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands.

Two dodecapeptides belonging to distinct classes of Src homology 3 (SH3) ligands and selected from biased phage display libraries were used to investigate interactions between a specificity pocket in the Src SH3 domain and ligant residues flanking the proline-rich core. The solution structures of c-Src SH3 complexed with these peptides were solved by NMR. In addition to proline-rich, polyproline type II helix-forming core, the class I and II ligands each possesses a flanking sequence that occupies a large pocket between the RT and n-Src loops of the SH3 domain. Structural and mutational analyses illustrate how the two classes of SH3 ligands exploit a specificity pocket on the receptor differently to increase binding affinity and specificity.

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.