The atmospheric dispersion of bacteria over long distances is an important facet of microbial ecology. Certain groups of dispersed bacteria can adapt to their new location and affect established ecosystems. Aeolian dust particles are known to be carriers of microbes but further research is needed to expand our understanding of this field of microbiology. Here we showed the potential of aeolian dust to global migration of bacterial cells. We demonstrated the presence of microbial cells on dust particles directly by bio-imaging. Bacterial abundance on dust particles declined from 10 5 to less than 10 3 cells/m 3 as the dust event subsided. Taxonomically diverse bacteria were identified by 16S rRNA gene sequencing and some of these bacteria retained growth potential. Our results confirm that bacteria can attach to aeolian dust particles and they have the potential to migrate globally during dust events and thus can contribute to the diversity of downwind ecosystems.
Although lipids are critical components of many cellular assemblies and biological pathways, accurate descriptions of their molecular structures remain difficult to obtain. Many benchtop characterization methods require arduous and time-consuming procedures, and multiple assays are required whenever a new structural feature is probed. Here, we describe a new mass-spectrometry-based workflow for enhanced structural lipidomics that, in a single experiment, can yield almost complete structural information for a given glycerophospholipid (GPL) species. This includes the lipid's sum (Brutto) composition from the accurate mass measured for the intact lipid ion and the characteristic headgroup fragment, the regioisomer composition from fragment ions unique to the sn-1 and sn-2 positions, and the positions of carbon-carbon double bonds in the lipid acyl chains. Here, lipid ions are fragmented using electron impact excitation of ions from organics (EIEIO)--a technique where the singly charged lipid ions are irradiated by an electron beam, producing diagnostic product ions. We have evaluated this methodology on various lipid standards, as well as on a biological extract, to demonstrate this new method's utility.
Uridine-disphosphate glucuronosyl transferase (UGT) enzymes catalyze the formation of glucuronide conjugates of Phase II metabolism. Methods for absolute quantification of UGT1A1 and UGT1A6 were previously established utilizing stable isotope peptide internal standards with LC-MS/MS. The current method expands upon this by quantifying eight UGT1A isoforms by nanobore HPLC coupled with a linear ion trap-time of flight mass spectrometer platform. Recombinant enzyme digests of each of the isoforms were used to determine assay linearity and detection limits. Enzyme expression level in human liver, kidney and intestinal microsomal protein was determined by extrapolation from spiked stable isotope standards. Intraday and Interday variability was <25% for each of the enzyme isoforms. Enzyme expression varied from 3 pmol/mg protein to 96 pmol/mg protein in liver and intestinal microsomal protein digests. Expression levels of UGT1A7, 1A8 and 1A10 were below detection limits (<1 pmol/mg protein) in HLMs. In kidney microsomes the expression of UGT1A3 was below detection limits, but levels of UGT1A4, 1A7, 1A9 and 1A10 protein were higher relative to liver, suggesting that renal glucuronidation could be a significant factor in renal elimination of glucuronide conjugates. This novel method allows quantification of all nine UGT1A isoforms, many previously not amenable to measurement with traditional methods such as immunologically based assays. Quantitative measurement of proteins involved in drug disposition, such as the UGTs, significantly improves the ability to evaluate and interpret in vitro and in vivo studies in drug development.
Background: Phosphatidic acid (PA) is involved in membrane dynamics. Results: PA-preferring phospholipase A 1 (PA-PLA 1 ) affects mitochondrial morphology in an activity-dependent manner. Gene disruption of PA-PLA 1 in mice causes sperm malformation due to mitochondrial organization defects. Conclusion: PA-PLA 1 regulates mitochondrial dynamics. Significance: We demonstrate an in vivo function of PA-PLA 1 and suggest a possible mechanism of PA regulation of the mitochondrial membrane.
We report on the first evidence of electron capture dissociation (ECD) in a radio frequency (rf) ion trap. Peptide ions, [substance P]2+, trapped in a two-dimensional, linear rf ion trap were cleaved by electrons injected along the central axis of the trap. Along the axis, the rf field component was zero and a magnetic field of 50 mT was applied. This electron injection scheme keeps the energy of the electrons below 1 eV, preventing them from heating by the rf field. The present ECD efficiency is approximately 4% by irradiation of electron current of 0.2 microA for 80 ms. ECD in rf traps may open high-throughput and low-cost ECD applications to obtain molecular structure information complementary to collision-induced dissociation.
A loss of balance between cell membrane-associated proteases and their inhibitors may underlie cancer invasion and metastasis. We analysed the roles of a membrane- associated serine protease inhibitor, HAI-1, in oral squamous cell carcinoma (OSCC). While membranous HAI-1 was widely observed in cancer cells of human OSCC tissues, this was significantly reduced at the infiltrative invasion front. In vitro, HAI-1 was detected in all eight OSCC cell lines examined, in which its cognate membrane protease, matriptase was also expressed. HAI-1 expression knock-down (KD) in OSCC lines, SAS and HSC-3, reduced the growth of both lines in vitro but significantly enhanced SAS tumourigenicity in vivo, which was accompanied by histological changes suggestive of the epithelial-mesenchymal transition. Both HAI-1-KD lines also exhibited significantly enhanced migratory capability, and membrane-associated but not truncated HAI-1 was required to rescue this phenotype. Other OSCC lines (HSC-2, Sa3, Ca9-22) also showed enhanced migration in response to HAI-1 KD. The enhanced migration is partly attributed to dysregulation of matriptase, as simultaneous matriptase KD alleviated the migration of HAI-1-KD cells. HAI-1 deficiency also altered the expression of CD24, S100A4, CCND2 and DUSP6, all of which are involved in tumour progression. While matriptase was involved in the increased CD24 expression associated with HAI-1 deficiency, the protease appeared to be not responsible for the altered expression of other genes. Therefore, a matriptase-independent mechanism for the invasiveness associated with HAI-1 KD is also present. Together, these observations suggest that HAI-1 has a crucial suppressive role in OSCC cell invasiveness.
Members of the intracellular phospholipase A1 family of proteins have been implicated in organelle biogenesis and membrane trafficking. The mammalian family comprises three members: phosphatidic acid-preferring phospholipase A1 (PA-PIA1)/DDHD1, p125/Sec23ip and KIAA0725p/DDHD2, all of which have a DDHD domain. PA-PLAI is mostly cytosolic, while KIAA0725p and p125 are more stably associated with the Golgi/endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and ER exit sites, respectively. Here we show that KIAAO725p and p125 are novel phosphoinositide-binding proteins. Deletion and mutational analyses of KIAAO725p suggested that a sterile alpha-motif (SAM), which is also present inp125, but not in cytosolic PA-PLAI, and the following DDHD domain comprise a minimal region for phosphatidylinositol 4-phosphate (Pl(4)P)-binding. A construct with mutations in the positively charged cluster of the SAM domain is defective in both phosphoinositide-binding and Golgi/ERGIC targeting. Consistent with the view that the Pl(4)P-binding is important for the membrane association of KIAA0725p, expression of phosphoinositide phosphatase Sacd reduces the association of expressed KIAAO725p with membranes. In addition, we show that deletion of the DDHD domain or introduction of point mutations at the conserved aspartate or histidine residues in the domain abolishes the phospholipase activity of KIAAO725p and PA-PLA1. Together, our results suggest that KIAAO725p is targeted to specific organelle membranes in a phosphoinositide-dependent manner, and that its SAM and DDHD domains are essential for its phosphoinositide-binding and phospholipase activity.
Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process.
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