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Platelet Microbicidal Protein 1: Structural Themes of a Multifunctional Antimicrobial Peptide. Nannette Y. Yount, 2004.Mammalian platelets release platelet microbicidal proteins (PMPs) as components of their antimicrobial armamentarium . The present studies defined the structure of PMP-1 and examined its structure-activity relationships . Amino acid sequencing and mass spectroscopy demonstrated that distinct N-terminal polymorphism variants of PMP-1 isolated from nonstimulated or thrombin-stimulated platelets arise from a single PMP-1 propeptide . Sequence data (NH2-[S]D1DPKE5SEGDL10HCVCV15KTTSL20 . . .) enabled cloning of PMP-1 from bone marrow and characterization of its full-length cDNA . PMP-1 is translated as a 106-amino-acid precursor and is processed to yield 73-residue (8,053 Da) and 72-residue (7,951-Da) variants . Searches with the BLAST program and sequence alignments demonstrated the homology of PMP-1 to members of the mammalian platelet factor 4 (PF-4) family of proteins . On the basis of phylogenetic relatedness, congruent sequence motifs, and predicted three-dimensional structures, PMP-1 shares the greatest homology with human PF-4 (hPF-4) . By integration of its structural and antimicrobial properties, these results establish the identity of PMP-1 as a novel rabbit analogue of the microbicidal chemokine (kinocidin) hPF-4 . These findings advance the hypothesis that stimuli in the setting of infection prompt platelets to release PF-4-class or related kinocidins, which have structures consistent with their likely multiple roles that bridge molecular and cellular mechanisms of antimicrobial host defense . Listeria monocytogenes Isolates from Foods and Humans Form Distinct but Overlapping Populations. Michael J. Gray, 2004.A total of 502 Listeria monocytogenes isolates from food and 492 from humans were subtyped by EcoRI ribotyping and PCR-restriction fragment length polymorphism analysis of the virulence gene hly . Isolates were further classified into genetic lineages based on subtyping results . Food isolates were obtained through a survey of selected ready-to-eat food products in Maryland and California in 2000 and 2001 . Human isolates comprised 42 isolates from invasive listeriosis cases reported in Maryland and California during 2000 and 2001 as well as an additional 450 isolates from cases that had occurred throughout the United States, predominantly from 1997 to 2001 . Assignment of isolates to lineages and to the majority of L . monocytogenes subtypes was significantly associated with the isolate source (food or human), although most subtypes and lineages included both human and food isolates . Some subtypes were also significantly associated with isolation from specific food types . Tissue culture plaque assay characterization of the 42 human isolates from Maryland and California and of 91 representative food isolates revealed significantly higher average infectivity and cell-to-cell spread for the human isolates, further supporting the hypothesis that food and human isolates form distinct populations . Combined analysis of subtype and cytopathogenicity data showed that strains classified into specific ribotypes previously linked to multiple human listeriosis outbreaks, as well as those classified into lineage I, are more common among human cases and generate larger plaques than other subtypes, suggesting that these subtypes may represent particularly virulent clonal groups . These data will provide a framework for prediction of the public health risk associated with specific L . monocytogenes subtypes . Microarray Transcription Analysis of Clinical Staphylococcus aureus Isolates Resistant to Vancomycin. Emmanuel Mongodin, 2003.The transcriptomes of vancomycin intermediate-resistance Staphylococcus aureus (VISA) clinical isolates HIP5827 and Mu50 (MIC = 8 µg/ml) were compared to those of highly vancomycin-resistant S . aureus (VRSA; MIC = 32 µg/ml) passage derivatives by microarray . There were 35 genes with increased transcription and 16 genes with decreased transcription in common between the two VRSAs compared to those of their VISA parents . Of the 35 genes with increased transcription, 15 involved purine biosynthesis or transport, and the regulator (purR) of the major purine biosynthetic operon (purE-purD) was mutant . We hypothesize that increased energy (ATP) is required to generate the thicker cell walls that characterize resistant mutants . A C35 Carotenoid Biosynthetic Pathway. Daisuke Umeno, 2003.Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C30 carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C35 backbone . The products of condensation of farnesyldiphosphate and GDP, C35 structures comprise 40 to 60% of total carotenoid accumulated . Carotene desaturases and carotene cyclases from C40 or C30 pathways accepted and converted the C35 substrate, thus creating a C35 carotenoid biosynthetic pathway in E . coli . Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described . This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures .
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