Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10⁶ DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10⁶ DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.     

Genome-wide patterns of expression in Drosophila pure species and hybrid males

One of the most fundamental questions for understanding the origin of species is why genes that function to cause fertility in a pure-species genetic background fail to produce fertility in a hybrid genetic background. A related question is why the sex that is most often sterile or inviable in hybrids is the heterogametic (usually male) sex. In this survey, we have examined the extent and nature of differences in gene expression between fertile adult males of two Drosophila species and sterile hybrid males produced from crosses between these species. Using oligonucleotide microarrays and real-time quantitative polymerase chain reaction, we have identified and confirmed that differences in gene expression exist between pure species and hybrid males, and many of these differences are quantitative rather than qualitative. Furthermore, genes that are expressed primarily or exclusively in males, including several involved in spermatogenesis, are disproportionately misexpressed in hybrids, suggesting a possible genetic cause for their sterility.     

Quantitative Proteomics of Presynaptic Mitochondria Reveal an Overexpression and Biological Relevance of Neuronal MitoNEET in Postnatal Brain Development

Although it has been recognized that energy metabolism and mitochondrial structure and functional activity in the immature brain differs from that of the adult, few studies have examined mitochondria specifically at the neuronal synapse during postnatal brain development. In this study, we examined the presynaptic mitochondrial proteome in mice at postnatal day 7 and 42, a period that involves the formation and maturation of synapses. Application of two independent quantitative proteomics approaches – SWATH‐MS and super‐SILAC – revealed a total of 40 proteins as significantly differentially expressed in the presynaptic mitochondria. In addition to elevated levels of proteins known to be involved in ATP metabolic processes, our results identified increased levels of mitoNEET (Cisd1), an iron‐sulfur containing protein that regulates mitochondrial bioenergetics. We found that mitoNEET overexpression plays a cell‐type specific role in ATP synthesis and in neuronal cells promotes ATP generation. The elevated ATP levels in SH‐SY5Y neuroblastoma cells were associated with increased mitochondrial membrane potential and a fragmented mitochondrial network, further supporting a role for mitoNEET as a key regulator of mitochondrial function.     

Kinetics and mechanism of oxygen-independent hydrocarbon hydroxylation by ethylbenzene dehydrogenase

Ethylbenzene dehydrogenase (EBDH) from the denitrifying bacterium Azoarcus sp. strain EbN1 (to be renamed Aromatoleum aromaticum) catalyzes the oxygen-independent, stereospecific hydroxylation of ethylbenzene to (S)-1-phenylethanol, the first known example of direct anaerobic oxidation of a nonactivated hydrocarbon. The enzyme is a trimeric molybdenum/iron-sulfur/heme protein of 155 kDa that is quickly inactivated in air in its reduced state. Enzyme activity can be coupled to ferricenium tetrafluoroborate, providing a convenient way for kinetic measurements. EBDH exhibits activity with a wide range of ethylbenzene analogues, which were analyzed for their kinetic parameters, stoichiometry, and formed products. The reactivity was correlated to the chemical structures by a quantitative structure-activity relationship (QSAR) model. On the basis of these results, quantum chemical calculations of DeltaG298 for formation of carbocations of the respective substrates were performed and used in reactivity analysis. A putative reaction mechanism is proposed on the basis of the experimental results and theoretical considerations. Finally, the enzyme reaction has been established in an electrochemical reactor, allowing sustained enzymatic reaction and potential technical applications of the enzyme.     

Adhesion of the genome-sequenced <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> IBB477 strain is mediated by specific molecular determinants

Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.     

Automatic particle selection: results of a comparative study

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.     

Patterns of antral follicular wave dynamics and accompanying endocrine changes in cyclic and seasonally anestrous ewes treated with exogenous ovine follicle-stimulating hormone during the inter-wave interval

In the ewe, ovarian follicular waves emerge every 4 to 5 days and are preceded by a peak in FSH secretion. It is unclear whether large antral follicle(s) in a wave suppress the growth of other smaller follicles during the inter-wave interval, as is seen in cattle. In this study, anestrous (n = 6; experiment 1) and cyclic (n = 5; experiment 2) Western white face ewes were given ovine FSH (oFSH) (0.5 microg/kg; two s.c. injections, 8 h apart) during the growth phase (based on ultrasonography) of a follicular wave (wave 1). Control ewes (n = 5 and 6, respectively) received vehicle. In oFSH-treated ewes, serum FSH concentrations reached a peak (P < 0.05) by 12 h after oFSH treatment, and this induced FSH peak did not differ (P > 0.05) from the endogenous FSH peaks. In all ewes, emergence of follicular waves 1 and 2 was seen (P > 0.05). However, in oFSH-treated ewes, an additional follicular wave emerged approximately 0.5 days after treatment: during the interwave interval of waves 1 and 2 without delaying the emergence of wave 2. The growth characteristics and serum estradiol concentrations did not differ (P > 0.05) between oFSH-induced waves and waves induced by endogenous FSH peaks. We concluded that, unlike in cattle, the largest follicle of a wave in sheep has limited direct effect on the growth of other follicles induced by exogenous oFSH. In addition, the largest follicle of a wave may possibly not influence the rhythmicity of follicular wave emergence, as it does in cattle.