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Seventy-eight Hereford-Angus crossbred heifers were injected intramuscularly twice with 6 mg of alfaprostolb in 6 ml of propylene glycol. On each representative day of a 20-day estrous cycle (estrus = Day 0), either three or four heifers received their first injection. The second injection was given 12 days after the first, regardless of the response to the first injection. Thirty-nine heifers were not treated. The first alfaprostol injection reduced serum progesterone to less than 1 ng/ml in all heifers injected after Day 4. A total of 79.5% (6278) of the heifers exhibited estrus by five days after the first injection. Average interval from injection to estrus was 63 hours. The second injection occurred on Days 6 through 16 for all but one heifer, with 75.6% (5978) falling on Days 8 through 11 of the estrous cycle. Estrus was detected in 93.6% (7378) of the heifers within five days after the second injection, with an average interval to estrus of 66 hours.Day of cycle at second injection did not affect the interval to estrus. Conception occurred in 79.4% (5873) of the heifers inseminated in the five days after the second injection. Occurrence of estrus and conception was no different in treated heifers after five days of the insemination period than in nontreated heifers after 21 days of the insemination period, where 94.9% (3739) were observed in estrus and 83.8% (3137) conceived. Overall percent conception for a 55-day insemination period was 89.7 (7078) for treated and 87.2 (3439) for nontreated heifers. Day of cycle at first or second injection did not affect conception after the second injection. Some signs of estrus were observed in 11 of the 16 heifers injected before Day 5.A second trial to determine if alfaprostol induced luteolysis early in the cycle was conducted. Twenty purebred Angus, Hereford, or Simmental heifers received either one or two injections of alfaprostol on either Day 1, 2, 3, or 4. Only five heifers showed any signs of estrus, and the three that were inseminated did not conceive. Subsequent cycle length indicated that luteolysis occurred in only one heifer.Data suggest that alfaprostol is an effective luteolytic agent in cyclic beef heifers after Day 4 and that two injections 12 days apart will effectively synchronize estrus in heifers when distributed throughout the cycle at the first injection without affecting conception rate.  相似文献   

3.
To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO3 transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO3 transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO3 currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO3-dependent pH recovery from a CO2-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO2/HCO3-free solution that was abolished by Cl removal from the bath. In the presence of CO2/HCO3, however, the outward current produced by D555E decreased only slightly after Cl removal. Starting from a Cl-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO2/HCO3. The substitution of Asp555 with an asparagine also produced a Cl current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO3. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp555 plays a role in HCO3 selectivity.The electrogenic Na/HCO3 cotransporter NBCe1 (SLC4A4) is one of the SLC4A gene family members transporting HCO3 across the plasma membrane (13). NBCe1 plays a role in transepithelial HCO3 movement and pHi regulation in many tissues (46). NBCe1 is responsible for HCO3 reabsorption in the proximal tubules of the kidney (7). The proximal tubule cells reclaim HCO3 from the lumen through a series of reactions involving titration of HCO3 by H+ secretion via the apical Na/H exchanger, production of CO2, and regeneration of HCO3 and H+ in the tubule cells. HCO3 then moves to the interstitium via the basolateral NBCe1. The essential feature driving this basolateral Na+/HCO3 exit is the stoichiometry of 1:3 Na+:HCO3, which makes the equilibrium potential for NBCe1 more positive than the resting membrane potential of the proximal tubule cells (8). The stoichiometry of 1Na+:1HCO3 or 1Na+:2HCO3 causes both ions to move into cells in other tissues such as pancreas, brain, and cardiovascular tissues (9, 10).Despite the importance of NBCe1 for basolateral HCO3 reabsorption in the proximal tubules, the mechanism of electrogenic Na/HCO3 transport via the transporter is not well understood. Ion movement depends on loading ions at their translocation or binding sites that likely reside within the membrane field at some distance from the bath solution (11). This implies that the transmembrane domains (TMs)2 of NBCe1 and amino acid residues within TMs play critical roles in ion transport.Sequence analysis of different SLC4A proteins shows similar hydropathy plots, predicting that these proteins share structural elements of transport function (12). Such similarities have facilitated structure/function studies to define molecular domains or motifs responsible for conferring Na/HCO3 transport of NBCe1. Abuladze et al. (13) performed a large scale mutagenesis on acidic and basic amino acids in non-TMs and found many residues affecting Na+-dependent base flux. McAlear et al. (14) identified amino acids in TM8 involving ion translocation. By a systematic approach of chimeric transporters between NBCe1 and the electroneutral Na/HCO3 cotransporter NBCn1 (SLC4A7) (15), we and our colleagues (16) demonstrated that electrogenic Na/HCO3 transport of NBCe1 requires interactions between the regions TM1–5 and TM6–13 of the protein. Zhu et al. (17) recently proposed TM1 as a domain lining the ion translocation pathway. On the other hand, Chang et al. (18) reported that the cytoplasmic N-terminal domain might contribute to HCO3 permeation.In the present study, we searched amino acid residues that are highly conserved among electrogenic Na/HCO3 transporters but not among electroneutral Na/HCO3 transporters and examined their role in electrogenic Na/HCO3 transport. Nine candidate residues in human renal NBCe1-A (5, 19) were selected and mutated by replacement with the amino acids at the corresponding sites of NBCn1. Mutant transporters were expressed in Xenopus oocytes and assessed via two-electrode voltage clamp. Our data show that Asp555 of NBCe1 plays an important role in HCO3 selectivity.  相似文献   

4.
The novel α1D L-type Ca2+ channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased α1D Ca2+ channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the α1 subunit of the α1D Ca2+ channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the α1 subunit of α1D Ca2+ channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the α1 subunit of α1D Ca2+ channel. These novel findings provide new insights into the autonomic regulation of the α1D Ca2+ channel in the heart.L-type Ca2+ channels are essential for the generation of normal cardiac rhythm, for induction of rhythm propagation through the atrioventricular node and for the contraction of the atrial and ventricular muscles (15). L-type Ca2+ channel is a multisubunit complex including α1, β and α2/δ subunits (57). The α1 subunit contains the voltage sensor, the selectivity filter, the ion conduction pore, and the binding sites for all known Ca2+ channel blockers (69). While α1C Ca2+ channel is expressed in the atria and ventricles of the heart (1013), expression of α1D Ca2+ channel is restricted to the sinoatrial (SA)2 and atrioventricular (AV) nodes, as well as in the atria, but not in the adult ventricles (2, 3, 10).Only recently it has been realized that α1D along with α1C Ca2+ channels contribute to L-type Ca2+ current (ICa-L) and they both play important but unique roles in the physiology/pathophysiology of the heart (69). Compared with α1C, α1D L-type Ca2+ channel activates at a more negative voltage range and shows slower current inactivation during depolarization (14, 15). These properties may allow α1D Ca2+ channel to play critical roles in SA and AV nodes function. Indeed, α1D Ca2+ channel knock-out mice exhibit significant SA dysfunction and various degrees of AV block (12, 1619).The modulation of α1C Ca2+ channel by cAMP-dependent PKA phosphorylation has been extensively studied, and the C terminus of α1 was identified as the site of the modulation (2022). Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a membrane-permeable cAMP analog, increased α1D Ca2+ channel activity using patch clamp studies (2). However, very little is known about potential PKA phosphorylation consensus motifs on the α1D Ca2+ channel. We therefore hypothesized that the C terminus of the α1 subunit of the α1D Ca2+ channel mediates its modulation by cAMP-dependent PKA pathway.  相似文献   

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The aim of the present study was to investigate the ganglioside expression of the highly metastatic murine lymphoreticular tumour cell line MDAY-D2. Cells were propagated under controlled pH conditions and oxygen supply in bioreactors of 1 and 7.5l volumes by repeated batch fermentation. Gangliosides were isolated from 2.7×1011 cells, purified by silica gel chromatography and separated into mono- and disialoganglioside fractions by preparative DEAE anion exchange high performance liquid chromatography. Individual gangliosides were obtained by preparative thin layer chromatography. Their structural features were established by immunostaining, fast atom bombardment and gas chromatography mass spectrometry. In addition to gangliosides of the GM1a-pathway (GM2, GM1a and GD1a) and GM1b (IV3Neu5Ac-GgOse4Cer) and GalNAc-GM1b of the GM1b-pathway, the dis8aloganglioside GD1 (IV3Neu5Ac, III6Neu5Ac-GgOse4Cer) was found in equal amounts compared to GD1a (IV3Neu5Ac, II3Neu5Ac-GgOse4Cer). All gangliosides were substituted with C24:0,24:1 and C16:0 fatty acids, sphingosine andN-acetylneuraminic acid as the sole sialic acid. Abbreviations: FAB-MS, fast atom bombardment-mass spectrometry; GC-MS, gas chromatography-mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac,N-acetylneuraminic acid; Neu5Gc,N-glycoloylneuraminic acid [57]. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [58] and the nomenclature of Svennerholm [59]. Gangliotriaosylceramide or GgOse3Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse4Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer gangliopentaosylceramide or GgOse5Cer, GalNAc1-4Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; GM2, II3Neu5Ac-GgOse3Cer; GM1a, II3Neu5Ac-GgOse4Cer; GM1b, IV3Neu5Ac-GgOse4Cer; GalNAc-GM1b, IV3Neu5Ac-GgOse5Cer; GD1a, IV3Neu5Ac, II3Neu5Ac-GgOse4Cer; GD1b, II3(Neu5Ac)2-GgOse4Cer; GD1 or GD1e, IV3Neu5Ac, III6Neu5AcGgOse4Cer; GD1e, IV3(Neu5Ac)2-GgOse4Cer; GT1b, IV3Neu5Ac, II3(Neu5Ac)2-GgOse4Cer.  相似文献   

7.
Allen  J. P.  Williams  J. C.  Graige  M. S.  Paddock  M. L.  Labahn  A.  Feher  G.  Okamura  M. Y. 《Photosynthesis research》1998,55(2-3):227-233
The direct charge recombination rates from the primary quinone, k AD (D+Q A DQA) and the secondary quinone, k BD (D+Q B DQB), in reaction centers from Rhodobacter sphaeroides were measured as a function of the free energy differences for the processes, G AD 0 and G BD 0 , respectively. Measurements were performed at 21 °C on a series of mutant reaction centers that have a wide range of dimer midpoint potentials and consequently a large variation in G AD 0 and G BD 0 . As –G AD 0 varied from 0.43 to 0.78 eV, k AD varied from 4.6 to 28.6 s–1. The corresponding values for the wild type are 0.52 eV and 8.9 s–1. Observation of the direct charge recombination rate k BD was achieved by substitution of the primary quinone with naphthoquinones in samples in which ubiquinone was present at the secondary quinone site, resulting specifically in an increase in the free energy of the D+Q A state relative to the D+QAQ B state. As –G BD 0 varied from 0.37 to 0.67 eV, k BD varied from 0.03 to 1.4 s–1. The corresponding values for the wild type are 0.46 eV and 0.2 s–1. A fit of the two sets of data to the Marcus theory for electron transfer yielded significantly different reorganization energies of 0.82 and 1.3 eV for k AD and k BD, respectively. In contrast, the fitted values for the coupling matrix element, or equivalently the maximum possible rate, were comparable (25 s–1) for the two charge recombination processes. These results are in accord with QB having more interactions with dipoles, from both the surrounding protein and bound water molecules, than QA and with the primary determinant of the maximal rate being the quinone-donor distance.  相似文献   

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The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular IP3-gated calcium (Ca2+) release channel and plays important roles in regulation of numerous Ca2+-dependent cellular responses. Many intracellular modulators and IP3R-binding proteins regulate the IP3R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP3R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP3R. The interaction was favored by the cytosolic Ca2+ concentration and it functionally inhibited IP3R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP3R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP3R-mediated Ca2+ signal through a direct interaction with IP3R in a cytosolic Ca2+-dependent manner.The inositol 1,4,5-trisphosphate (IP3)3 receptor (IP3R) consisting of three subtypes, IP3R1, IP3R2, and IP3R3, is a tetrameric intracellular IP3-gated calcium (Ca2+) release channel localized at the endoplasmic reticulum (ER) with its NH2 terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP3Rs are composed of five functional domains. The long NH2-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP3-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP3R channel opening (2, 3). Ca2+ release activity of the IP3R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca2+), protein kinases, and IP3R-binding proteins (2, 4), and the tight regulation of IP3R channel activity by these factors generates various spatial and temporal intracellular Ca2+ patterns such as Ca2+ spikes and Ca2+ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP3R. A, schematic of ER residential IP3R. The CTT of IP3R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP3R1 in vitro. GST and GST-IP3R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP3R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP3R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP3R1. F and G, both GIT1 and GIT2 bind to all three IP3R subtypes. HeLa cells coexpressing GFP-fused IP3R1, IP3R2, or IP3R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP3R-mediated Ca2+ signaling is a pro-apoptotic regulator during apoptosis. Ca2+ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca2+ released from the IP3Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-XL (12), have been reported to interact with the internal coupling domain and/or the CTT of IP3R and enhance the Ca2+-release activity of IP3Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP3R-binding proteins that bind to the CTT of IP3R and inhibit apoptosis by regulation of IP3R-mediated Ca2+ signal.  相似文献   

10.
As a second messenger, H2O2 generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H2O2 signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H2O2 generation, inhibition of inward K+ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H2O2 signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H2O2, is essential to plant growth and development in response to stresses,14 and involves activation of various signaling events, among which are the MAP kinase cascades.13,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,4,68 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H2O2 generation.6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H2O2 signaling regardless of SA mimicking ABA in regulating stomatal closure.2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H2O2 generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.1113 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,1416 Pisum sativum,17 Medicago sativa18 and tobacco.19 The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,20 tobacco tissue21 and suspension-cultured Oryza sativa cells.22 Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H2O2 signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H2O2 generation and decreasing K+ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H2O2 signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H2O2 signaling,23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase23,24 is not always identical to that of MEK1/28,25 in ABA-induced H2O2 signaling of Vicia guard cells. For example, H2O2- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H2O2, the effects of both ABA- and H2O2-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H2O2-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H2O2-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.26 Similar to this, SB203580 improves H2O2-inhibited inward K+ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H2O2 signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,1,2,8,23,24 and the ABA and H2O2 pathways diverge further downstream in their actions on the K+ channels and, thus, on stomatal control. Other differences in action between ABA and H2O2 are known. For example, Köhler et al. (2001) reported that H2O2 inhibited the K+ outward rectifier in guard cells shows that H2O2 does not mimic ABA action on guard cell ion channels as it acts on the K+ outward rectifier in a manner entirely contrary to that of ABA.27Open in a separate windowFigure 2Effect of SB203580 on ABA- and H2O2-inhibited inward K+ currents. The experimental procedure and data analysis are according to the previous publication.24 SB203580 directs ABA- and H2O2-inactivated inward K+ currents across plasma membrane of Vicia guard cells. Here the inward K+ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H2O2 signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,14 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H2O2 signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.  相似文献   

11.
Butterbach-Bahl  K.  Rothe  A.  Papen  H. 《Plant and Soil》2002,240(1):91-103
Complete annual cycles of N2O and CH4 flux in forest soils at a beech and at a spruce site at the Höglwald Forest were followed in 1997 by use of fully automatic measuring systems. In order to test if on a microsite scale differences in the magnitude of trace gas exchange between e.g. areas in direct vicinity of stems and areas in the interstem region at both sites exist, tree chambers and gradient chambers were installed in addition to the already existing interstem chambers at our sites. N2O fluxes were in a range of –4.6–473.3 g N2O-N m–2 h–1 at the beech site and in a range of –3.7–167.2 g N2O-N m–2 h–1 at the spruce site, respectively. Highest N2O emissions were observed during and at the end of a prolonged frost period, thereby further supporting previous findings that frost periods are of crucial importance for controlling annual N2O losses from temperate forests. Fluxes of CH4 were in a range of +10.4––194.0 g CH4 m–2 h–1 at the beech site and in a range of –4.4––83.5 g CH4 m–2 h–1 at the spruce site. In general, both N2O-fluxes as well as CH4-fluxes were higher at the beech site. On a microsite scale, N2O and CH4 fluxes at the beech site were highest within the stem area (annual mean: 49.6±3.3 g N2O-N m–2 h–1; –77.2±3.1 g CH4 m–2 h–1), and significantly lower within interstem areas (18.5±1.4 g N2O-N m–2 h–1; –60.2±1.8 g CH4 m–2 h–1). Significantly higher values of total N, C and pH in the organic layer, as well as increased soil moisture, especially in spring, in the stem areas, are likely to contribute to the higher N2O fluxes within the stem area of the beech. Also for the spruce site, such differences in trace gas fluxes could be demonstrated to exist (mean annual N2O emission within (a) stem areas: 9.7±0.9 g N2O-N m–2 h–1 and (b) interstem areas: 6.2±0.6 g N2O-N m–2 h–1; mean annual CH4 uptake within (a) stem areas: –26.1±0.6 g CH4 m–2 h–1 and (b) interstem areas: –38.4±0.8 g CH4 m–2 h–1), though they were not as pronounced as at the beech site.  相似文献   

12.
Desulfovibrio vulgaris (Marburg) was grown on H2 plus sulfate and H2 plus thiosulfate as the sole energy sources and acetate plus CO2 as the sole carbon sources. Conditions are described under which the bacteria grew exponentially. Specific growth rates () and molar growth yields (Y) at different pH were determined. and Y were found to be strongly dependent on the pH. Highest growth rates and molar growth yields were observed for growth on H2 plus sulfate at pH 6.5 (=0.15h-1; Y SO 4 2- =8.3g·mol-1) and for growth on H2 plus thiosulfate at pH 6.8 (=0.21h-1; Y S 2O 3 2 =16.9g·mol-1).The growth yields were found to increase with increasing growth rates: plots of 1/Y versus 1/ were linear. Via extrapolation to infinite growth rates a Y SO4 2- /max of 12.2g·mol-1 and a YS2O 3 2- /max of 33.5g·mol-1 was obtained.The growth yield data are interpred to indicate that dissimilatory sulfate reduction to sulfide is associated with a net synthesis of 1 mol of ATP and that near to 3 mol of ATP are formed during dissimilatory sulfite reduction to sulfide.  相似文献   

13.
Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.2,3 This defense strategy has been known as the hypersensitive response (HR).4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.6Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.2 Although ROS have been identified downstream of LCBs in the route towards PCD,1 the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.7Similar to what is observed in pathogen defense,3 increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.1,2,911An early production of ROS has been linked to an increase of LCBs. For example, an H2O2 burst is found in tobacco cells after 2–20 min of sphinganine supplementation,12 and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H2O2 were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.1 However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,15 or to intracellular ROS that diffuse outwards.16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references 28 and 29. DPI was used at 50 µM. (B) H2O2 formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H2O2 according to Thordal-Christensen et al.30It has been suggested that the H2O2 burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.12 It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.9 In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.15 This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.2 All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H2O2 formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H2O2 with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis18 and the mutant has a FB1-resistant phenotype.2 These results indicate that mutations in the LCB11 and LCB2a2 genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H2O2. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.3 In this condition, we clearly detected H2O2 formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.2 Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H2O2 formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.2Open in a separate windowFigure 2Conditions of LCBs elevation produce H2O2 formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H2O2 formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 108 UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H2O2 formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl2 and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.2 Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H2O2 produce alterations in mitochondrial morphology.2022 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.2224It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.26 At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD27 as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.  相似文献   

14.
Summary Relaxation times of 13C carbons of uniformly 13C/15N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T1, T2 and the heteronuclear NOE of 13C in uniformly 13C/15N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T1 and the heteronuclear NOE were similar to those of the corresponding 15N experiments published previously. The determination of T2 for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T2 evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T2 evolution period were simulated in order to optimize the bandwidth for which reliable T2 relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual 1H–13C cross peaks, in a series of (1H, 13C)-ct-HSQC spectra with a modified CPMG sequence as well as a T1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.  相似文献   

15.
16.
YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-14C]galactose andd-[1-14C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside. As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone. No cross-reaction with GM1b or GgOse4Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse3Cer or gangliotriaosylceramide or asialo GM2, GalNAc1-4Gal1-4GlcCer; GgOse4Cer or gangliotetraosylceramide or asialo GM1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse5Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II3NeuAc-GgOse4Cer or GM1; IV3NeuAcGgOse4Cer or GM1b; IV3NeuAc-GgOse5Cer or GalNAc-GM1b; IV3NeuAc, II3NeuAc-GgOse4Cer or GD1a; II3(NeuAc)2-GgOse4Cer or GD1b; IV3(NeuAc)2-GgOse4Cer or GD1c; IV3NeuAc,III6NeuAc-GgOse4Cer or GD1a; IV3NeuAc,II3(NeuAc)2-GgOse4Cer or GT1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).  相似文献   

17.
The following neolacto glycolipids were identified and their developmental expression was studied in the rat cerebral cortex and cerebellum: Fuc1-3IIInLcOse4Cer,Fuc1-3VnLcOse6Cer and (Fuc)21-3III,3VnLcOse6Cer, as well as acidic glycolipids, NeuAc2-3IVnLcOse4Cer [nLM1], (NeuAc)22-3IVnLcOse4Cer [nLD1],O-acetyl (NeuAc)22-3IVnLcOse4Cer [OAc-nLD1] and their higher neolactosaminyl homologues NeuAc2-3VInLcOse6Cer [nHM1] and (NeuAc)22-3VInLcOse6Cer [nHD1]. These glycolipids were expressed in the cerebral cortex only during embryonic stages and disappeared postnatally. This loss was ascribed to the down regulation of the synthesis of the key precursor LcOse3Cer which is synthesized by the enzyme lactosylceramide:N-acetylglucosaminyl transferase. On the other hand in the cerebellum, these glycolipids increased with postnatal development due to increasing availability of LcOse3Cer. In the cerebellum, only nLM1 and fucosyl-neolactoglycolipids declined after postnatal day 10–15, perhaps due to regulation by other glycosyltransferases. Also, in the cerebellum, nLD1 and nHD1 were shown to be specifically associated with Purkinje cells and their dendrites in the molecular layer and with their axon terminals in the deep cerebellar nuclei, similar to other neolactoglycolipids shown previously.  相似文献   

18.
The three-substituted dipyridyl ligand bis(3-pyridylmethyl)sulfide (L1) was prepared by the reaction of 3-(chloromethyl)pyridine hydrochloride with thioacetamide under basic conditions. L1 was reacted with CuI to give complexes with 1:2 and 1:1 molar ratios. Crystal structures of [(CuI)2(L1)] (1) and [CuI(L1)] (2) were determined. In complex 1 the CuI species formed a one-dimensional staircase polymer to which L1 was bound in a side-by-side fashion with π-π interactions between the ligands on each side. Complex 2 consisted of a one-dimensional ribbon polymer of metallomacrocycles formed from two L1 ligands bridging Cu2I2 dimers which were fused within the macrocyclic ring. The analogous disulfide ligand bis(3-pyridylmethyl)disulfide (L2) was prepared by oxidation of the corresponding thiol 3-(sulfanylmethyl)pyridine. L2 was reacted with CuI in 1:2 and 1:1 molar ratios and products isolated but only the 1:1 product was able to be crystallised. The crystal structure of [CuI(L2)] (3) consisted of a one-dimensional ribbon polymer of metallomacrocycles formed from two L2 ligands linked through Cu2I2 dimers. The difference in the metallomacrocycle linking between the related structures 2 and 3 was attributed to the difference in ligand conformation.  相似文献   

19.
20.
Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log10 and 6 log10 CFU g1.Bovines are a reservoir for verotoxigenic Escherichia coli O157:H7 and Campylobacter jejuni, pathogenic microorganisms responsible for severe human gastrointestinal disease (5, 12). Qualitative and quantitative detection of these organisms in bovine feces is essential for evaluating risk to human health. Real-time PCR (quantitative PCR [qPCR]) assays have been developed to detect and quantify both E. coli O157:H7 and C. jejuni bacteria by using DNA directly extracted from animal feces (20, 22). Analysis of DNA extracted from bovine feces can generate a high level of correlation between the actual target cell density and the PCR signal (7, 8). However, the detection of E. coli O157:H7 and C. jejuni by direct DNA extraction is less sensitive and more variable than detection by procedures based on a preliminary enrichment step (e.g., laboratory culture) (7, 9, 16, 20). We explored the potential of lyophilization for improving overall detection by qPCR through increasing the amount of bovine fecal material available for DNA extraction.Four sets of five fresh bovine fecal samples were collected, and each sample was divided into four equal portions. Samples were seeded with either (i) E. coli O157:H7 (strain NZRM 3614) grown for 18 h at 37°C in tryptic soy broth (BD, Sparks, MD) or (ii) C. jejuni (strain NZRM 1958) grown for 48 h at 42°C in Exeter broth (11) to obtain the following concentrations: set 1, 0 CFU g1 (unseeded) and 3.5 log10, 4.5 log10, and 5.5 log10 CFU of E. coli O157:H7 g1, and set 2, 0 CFU g1 (unseeded) to 5.2 log10 CFU of E. coli O157:H7 g1. Set 3 and 4 concentrations varied from 0 CFU g1 (unseeded) to 6.4 log10 C. jejuni CFU g1. DNA was either extracted directly from fresh samples or extracted from samples after lyophilization. Lyophilization involved mixing of prepared fecal samples in phosphate-buffered saline (145 mM NaCl, 59 mM Na2HPO4, 8 mM KH2PO4, pH 7.5) at a ratio of 1:10 (wt/vol), homogenization with a lab blender model 400 (Seward Medical, London, United Kingdom), cooling to −35°C, and concentration using a 1015GP lyophilizer (Cuddon Ltd., Blenheim, New Zealand). Total DNA was extracted from 0.2 g of a fresh or lyophilized fecal sample by using a QIAamp DNA stool minikit (Qiagen Inc., Mississauga, Canada). DNA was amplified using either a TaqMan E. coli O157:H7 detection kit (Applied Biosystems, Foster City, CA) or mapA primers and a corresponding probe (1). Amplification and fluorescence data were collected with optical-grade 96-well plates by using a TaqMan 7300 PCR system (Applied Biosystems). For each DNA sample, a mean threshold cycle (CT) value for triplicate qPCR runs was calculated. When no CT value was obtained, an arbitrary CT value of 40 was assigned. All data were reported as equivalent concentrations in fresh feces. Significance levels were determined by one-way analysis of variance. The relationship between the log10 numbers of CFU g1 fresh feces (viable-cell counts) and CT values was analyzed using GenStat software (version 10.2.0.175; VSN International, Oxford, United Kingdom). Confidence intervals were obtained using the software program Flexi (21).Lyophilized samples were associated with significantly improved sensitivity (P < 0.001) at seeding levels of 4.5 and 5.5 log10 E. coli O157:H7 CFU g1 (Table (Table1).1). At 3.5 log10 CFU g1, the rate of E. coli O157:H7 detection was also higher, with all lyophilized samples producing a CT value of <40 (Table (Table1).1). Individual CT values for the three qPCR amplification runs were sufficiently similar to allow averaging (P > 0.05). Regression analysis of the averaged set 2 and 3 data (Fig. (Fig.1)1) demonstrated that the detection of both E. coli O157:H7 and C. jejuni was linear for seeding levels ranging from ca. 2 log10 to 6 log10 CFU g1 fresh feces. The range of concentrations used reflects the reported range of concentrations of these bacteria in feces (i.e., 0 to 6 log10 CFU g1) as determined by conventional culture (3, 4, 18, 19). The high coefficients of correlation for the relationships between the log10 numbers of CFU g1 feces and the CT values indicated the specific amplification of the target DNA. The reproducibility of detection of E. coli O157:H7 was reduced at the lowest seeding concentration (i.e., 2.2 log10 CFU g1 feces), with 75% of the samples giving a CT value of <40. The limit for 100% successful detection after lyophilization was 2.9 log10 E. coli O157:H7 CFU g1. The detection of C. jejuni by qPCR varied between sets. For set 3, 100% reproducibility occurred at 2.2 log10 C. jejuni CFU g1. For set 4, satisfactory detection was obtained only after dilution of the DNA extract prior to qPCR. Despite this requirement for dilution, C. jejuni was still detected in 80% of the samples of set 4 seeded at a density of 2.2 log10 C. jejuni CFU g1.Open in a separate windowFIG. 1.Ranges of quantification of E. coli O157:H7 (A) and C. jejuni (B) bacteria obtained from lyophilized fecal samples by real-time PCR. Each point represents the average CT value for triplicate runs of one fecal sample at one seeding concentration. The hatched areas represent the 95% confidence intervals.

TABLE 1.

Difference in CT values obtained for real-time PCR detection of E. coli O157:H7 in seeded fecal samples (n = 5) with and without lyophilization
Seeding level (log10 CFU g−1 fresh feces) or statusAverage CT value (range)
Without lyophilizationWith lyophilization
5.531.50 (31.02-32.18)28.34 (28.04-29.03)
4.534.79 (33.43-35.75)31.33 (31.01-31.89)
3.535.45a33.52 (33.21-33.87)
Unseeded>40>40
Open in a separate windowaOnly one fecal sample gave a CT value of <40.Overall, the removal of water by lyophilization provided an approximately 10-fold increase in the amount of fecal material used. Consequently, the test sensitivity was 10-fold greater than that reported previously (17, 7). Lyophilization of feces has been reported to be useful for PCR-based studies of pigs (14), and our results indicate a useful role for the quantification of E. coli O157:H7 bacteria in cattle feces. Indeed, the slopes and the linear regression coefficients for the qPCR signal (CT values) and the known concentrations of microbial pathogen cells in the feces are in agreement with published values (2). Our methodology shows a lower limit of C. jejuni quantification by qPCR (ca. 2 log10 CFU g1 in seeded fresh feces) than that reported previously (8), demonstrating the usefulness of lyophilization to improve detection and quantification of bacteria in feces.In our study, the accurate detection of C. jejuni after DNA extraction from lyophilized feces was adversely affected for some samples. Interference due to partial removal of PCR inhibitors after DNA extraction using the QIAamp DNA stool minikit has been reported by other workers (10, 15). For lyophilized samples, the inhibition was successfully overcome by dilution of DNA. Recent reports confirmed the importance of diluting DNA (up to 3 log) to increase the accuracy of detection by real-time PCR (6, 13). Lyophilization presents the advantage that lyophilized material can be stored for long periods at room temperature, is easy to transport, and can also be used for complementary chemical analysis.  相似文献   

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