Acidic O-specific polysaccharide containing D-glucose, D-glucuronic acid, L-fucose, and 2-acetamido-2-deoxy-D-glucose was obtained by mild acid degradation of lipopolysaccharide from Providencia alcalifaciens O46. The following structure of the hexasaccharide repeating unit of the O-specific polysaccharide was established using methylation analysis along with 1H and 13C NMR spectroscopy, including 2D 1H, 1H-COSY, TOCSY, ROESY, 1H, 13C-HSQC, and HMQC-TOCSY experiments:
Physiological processes are often activated by reactive oxygen species (ROS), such as the superoxide anion (O2–) and nitric oxide (NO) produced by cells. We studied the interactions between NO and O2–, and their generators (NO synthase, NOS, and a still elusive oxidase), in human spermatozoa during capacitation (transformations needed for acquisition of fertility). Albumin, fetal cord serum ultrafiltrate, and L-arginine triggered capacitation and ROS generation (NO and O2–) and superoxide dismutase (SOD) and NOS inhibitors prevented all these effects. Surprisingly, capacitation due to exogenous NO (or O2–) was also blocked by SOD (or NOS inhibitors). Probes used were proven specific and innocuous on spermatozoa. Whereas O2– was needed only for 30 min, the continuous NO generation was essential for hours. Capacitation caused a time-dependent increase in protein tyrosine nitration that was prevented by SOD and NOS inhibitors, suggesting that O2– and NO· also act via the formation of ONOO–. Spermatozoa treated with NO (or O2–) initiated a dose-dependent O2– (or NO) production, providing, for the first time in cells, a strong evidence for a two-sided ROS-induced ROS generation. Data presented show a close interaction between NO and O2– and their generators during sperm capacitation. 相似文献
Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar 15N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought.Key words: hydraulic lift, nitrogen, phosphorus, microbial abundance, mycorrhizal hyphae, QuercusIn environments that experience seasonal or extended drought, plant productivity, resource partitioning, and competition are limited by the availability of water and mineral nutrients. One mechanism that is important to whole plant water balance in these environments is hydraulic lift (HL), a passive process driven by gradients in water potential among soils layers. Soil water is transported upwards from deep moist soils and released into the nutrient-rich upper soil layers by root systems accessing both deep and shallow soil layers.1 HL water may improve the lifespan and activity of fine roots in a wide variety of plant life forms.2Hydraulic lift may also have a second ecological function in facilitating plant nutrient acquisition.2 It been hypothesized that HL water could enhance the supply of nutrients to roots through mass flow or diffusion,3 or trigger episodes of soil biotic activity such as microbe-mediated nutrient transformations4,5 that are analogous to the increased inflow of nitrogen (N) into roots and flushes of carbon (C) and N mineralization respectively that follow precipitation events.4,6 However, few data currently exist with which to test these possibilities.Hydraulically lifted water also sustains mycorrhizal fungi,7,8 a mutualism that enhances the acquisition of water and mineral nutrients in many terrestrial plant species. Mycorrhizal fungal hyphae provide comprehensive exploration and rapid access to small-scale or temporary nutrient flushes that may not be available to plant roots.9 This resource flow has often been assumed to be a unidirectional flux whereby resources are moved from source (soil) into the sink (plant) by the fungal hyphae. However, there is now evidence to suggest that the physiological plasticity of the peripheral extramatrical hyphae, and in particular the hyphal tips, permits the exudation, and subsequent reabsorption, of water and solutes.10,11 Laboratory experiments using pure cultures have demonstrated that water may be exuded from the hyphal tips, especially in fungal species with hydrophobic hyphae, along with a variety of organic molecules, such as free amino acids.10–13 At the same time, water, mobile minerals, amino acids and other low-molecular weight metabolites may be selectively and actively reabsorbed by mycorrhizal fungal hyphae.11 However, quantitative data on the environmental impact of hyphal exudation and reabsorption is still largely lacking.We ask: could the diel cycle of water availability created by HL produce a water efflux from hyphal tips and if so, would this be sufficient to impact biogeochemical processes? Is there also an opposite rhythm driven by plant transpiration so that any resultant soil solution is pulled towards hyphal tips and consequently, the host plant? By imposing drought on seedlings of Quercus agrifolia Nee (coast live oak; Fagaceae) grown in mesocosms (Fig. 1), we identified a composite of feedbacks that could influence nutrient capture with HL (Fig. 2). Our analyses provide support for the key predictions of the HL-nutrient cycling scenario including the efflux of HL water from the extramatrical hyphae (Fig. 3), moistening of soil organic matter (Figs. 3 and and4),4), and the maintenance of soil microbial activity and nutrient capture (N, P; Open in a separate windowFigure 1Quercus mesocosms demonstrating the plant, root, and hyphal compartments. Details of soil conditions, plant inoculation protocol, mycorrhizal fungi and dye injection methods are detailed in previous work (ref. 7) Point 1 (tap root compartment) denotes the region in which fluorescent tracer dyes were injected into the mesocosm at dusk to track the path of HL water. Point 2 (hyphal chamber) denotes spots adjacent to or distant from the mesh screen into which a small volume (200 µl) of fluorescent and 15N tracers (99% as 15NH415NO3) were injected at dawn to measure water and nutrient uptake by the external hyphae.Open in a separate windowFigure 2Path analysis of the influence of different soil and mycorrhizal factors on nutrient capture with HL, and resultant model showing the significant path coefficients among variables in the Q. agrifolia mesocosms. Lines with a single arrow denote possible cause-effect relationships. The partial correlation coefficients adjacent to each line indicate the strength of the association between the individual factors. Thick lines are statistically significant (p < 0.05) whereas thin lines indicate no significant relationship between parameters (p > 0.05) and only significant coefficients are given (p < 0.05).Open in a separate windowFigure 3Fluorescently-labeled structures recovered from the hyphal chamber of Quercus microcosms following 80 days of soil drying and with nocturnal hydraulic lift. Yellow-green fluorescence indicates samples labeled with Lucifer yellow CH (LYCH), blue fluorescence denotes samples labeled with Cascade blue (CB) hydrazide. (A) CB-labeled leaf litter from the soil and (B) soil particle; (C) LYCH-labeled root fragment in the soil mixture with adherent extramatrical hyphae; (D) LYCH tracer dye fluorescence in labeled extramatrical hyphae and in efflux (arrow) from the hyphal tip onto organic matter; (E and F) external hyphae filled with LYCH (influx; arrow) and (G) background fluorescence in non-labeled extramatrical hyphae.Open in a separate windowFigure 4Measurements of hyphal efflux and influx based on the quantitative analysis of LYCH fluorescence intensity in soil solution. Fluorescent intensity values were converted to LYCH concentration using a standard curve generated for the dye since fluorescent intensity correlates with the number of fluorescent molecules in solution. Influx is the uptake of LYCH by hyphae as driven by plant transpiration demands (day), and measured efflux is the passive loss of LYCH from hyphae into the surrounding soil during HL (night). Vertical bars indicate the standard error of the means.
Table 1
Summary of soil, microbial, mycorrhizal and plant parameters in plant or hyphal compartments
Compartment and Location
Trait
Plant
Hyphal (Near Mesh)
Hyphal (Away from Mesh)
γs Dawn (MPa)
-4.19 (0.31)b
-2.04 (0.66)a
-2.09 (0.31)a
γs Dusk (MPa)
-20.3 (2.10)b
-2.55 (0.49)a
-2.09 (0.30)a
Phosphatase activity (µg pNP g-1 hr-1)
346 (41)b
1289 (38)a
1128 (33)a
Microbial abundance (colonies g-1 soil x 106)
2.55 (0.28)b
4.72 (1.21)a
3.54 (0.37)a
Total hyphal length (AMF + EM; m g-1 soil)
29 (13)b
235 (45)a
208 (52)a
Live hyphal length (dye-labeled AMF + EM hyphae; m g-1 soil)
29 (3.5) b
75 (0.3)a
69 (2.1)a
*Abundance of microbial genes:
16s rRNA
++
++
++
nirK
+
+
+
nirS
nd
nd
nd
amoA
++
++
++
§Percentage of 15N incorporated into plant or fungal biomass
Old leaves 0.10
Hyphae 4.34
Hyphae 5.70
New leaves 5.74
Fine roots 1.42
Open in a separate windowWithin each row, mean values with the same letter do not differ significantly at p < 0.05.*Microbial genes: + detected in soil; ++ abundant in soil; nd, not detected in sample.§Percentage of 15N uptake based on two-source mixing-model of δ15N (‰) in plant and hyphal material following the spot application of 15NH415NO3 to the hyphal compartment. 相似文献
Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.Key words: prion diseases, prion interference, prion strainsPrion diseases are fatal neurodegenerative diseases that are caused by an abnormal isoform of the prion protein, PrPSc.1 Prion strains are hypothesized to be encoded by strain-specific conformations of PrPSc resulting in strain-specific differences in clinical signs, incubation periods and neuropathology.2–7 However, a universally agreed upon definition of prion strains does not exist. Interspecies transmission and adaptation of prions to a new host species leads to the emergence of a dominant prion strain, which can be due to selection of strains from a mixture present in the inoculum, or produced upon interspecies transmission.8,9 Prion strains, when present in the same host, can interfere with each other.Prion interference was first described in mice where a long incubation period strain 22C extended the incubation period of a short incubation period strain 22A following intracerebral inoculation.10 Interference between other prion strains has been described in mice and hamsters using rodent-adapted strains of scrapie, TME, Creutzfeldt-Jacob disease and Gerstmannn-Sträussler-Scheinker syndrome following intracerebral, intraperitoneal, intravenous and sciatic nerve routes of inoculation.10–15 We previously demonstrated the detection of PrPSc from the long incubation period DY TME agent correlated with its ability to extend the incubation period or completely block the superinfecting short incubation period HY TME agent from causing disease and results in a reduction of HY PrPSc levels following sciatic nerve inoculation.12 However, it is not known if a single long incubation period agent (e.g., DY TME) can interfere with more than one short incubation period agent or if interference can occur by a natural route of infection.To examine the question if one long incubation period agent can extend the incubation period of additional short incubation period agents, hamsters were first inoculated in the sciatic nerve with the DY TME agent 120 days prior to superinfection with the short-incubation period agents HY TME, 263K scrapie and HaCWD.16–18 The HY TME and 263K scrapie agents have been biologically cloned and have distinct PrPSc properties.19,20 The HaCWD agent used in this study is seventh hamster passage that has not been biologically cloned and therefore will be referred to as a prion isolate. Sciatic nerve inoculations were performed as previously described.11,12 Briefly, hamsters were inoculated with 103.0 i.c. LD50 of the DY TME agent or equal volume (2 µl of a 1% w/v brain homogenate) of uninfected brain homogenate 120 days prior to superinfection of the same sciatic nerve with either 104.6 i.c. LD50 of the HY TME agent, 105.2 i.c. LD50 of the HaCWD agent or 104.6 i.c. LD50/g 263K scrapie agent (Bartz J, unpublished data).16,18,21 Animals were observed three times per week for the onset of clinical signs of HY TME, 263K and HaCWD based on the presence of ataxia and hyperexcitability, while the clinical diagnosis of DY TME was based on the appearance of progressive lethargy.16–18 The incubation period was calculated as the number of days between the onset of clinical signs of the agent strain that caused disease and the inoculation of that strain. The Student''s t-test was used to compare incubation periods.12 We found that sciatic nerve inoculation of both the HaCWD agent and 263K scrapie agent caused disease with a similar incubation period to animals infected with the HY TME agent (12 In hamsters inoculated with the DY TME agent 120 days prior to superinfection with the HaCWD or 263K agents, the animals developed clinical signs of DY TME with an incubation period that was not different from the DY TME agent control group (12 The PrPSc migration properties were consistent with the clinical diagnosis and all co-infected animals had PrPSc that migrated similar to PrPSc from the DY TME agent infected control animal (Fig. 1, lanes 1–10). This data indicates that the DY TME agent can interfere with more than one isolate and that interference in the CNS may be a more generalized phenomenon of prion strains.Open in a separate windowFigure 1The strain-specific properties of PrPSc correspond to the clinical diagnosis of disease. Western blot analysis of 250 µg brain equivalents of proteinase K digested brain homogenate from prion-infected hamsters following intracerebral (i.c.), sciatic nerve (i.sc.) or per os inoculation with either the HY TME (HY), DY TME (DY), 263K scrapie (263K), hamster-adapted CWD (CWD) agents or mock-infected (UN). The unglycoyslated PrPSc glycoform of HY TME, 263K scrapie and hamster-adapted CWD migrates at 21 kDa. The unglycosylated PrPSc glycoform of DY PrPSc migrates at 19 kDa. Migration of 19 and 21 kDa PrPSc are indicated by the arrows on the left of the figure. n.a., not applicable.
Table 1
Clinical signs and incubation periods of hamsters inoculated in the sciatic nerve with either the HY TME, HaCWD or 263K scrapie agents, or co-infected with the DY TME agent 120 days prior to superinfection of hamsters with the HY TME, HaCWD or 263K agents
Onset of clinical signs
First inoculation
Interval between inoculations
Second inoculation
Clinical signs
PrP-res migration
A/Ia
After 1st inoculation
After 2nd inoculation
Mock
120 days
HY TME
HY TME
21 kDa
5/5
n.a.
72 ± 3b
Mock
120 days
HaCWD
HaCWD
21 kDa
5/5
n.a.
73 ± 3
Mock
120 days
263K
263K
21 kDa
5/5
n.a.
72 ± 3
DY TME
120 days
Mock
DY TME
19 kDa
4/4
224 ± 2
n.a.
DY TME
120 days
HY TME
DY TME
19 kDa
5/5
222 ± 2c
102 ± 2
DY TME
120 days
HaCWD
DY TME
19 kDa
5/5
223 ± 3c
103 ± 3
DY TME
120 days
263K
DY TME
19 kDa
5/5
222 ± 2c
102 ± 2
Open in a separate windowaNumber affected/number inoculated;bAverage days postinfection ± standard deviation;cIncubation period similar compared to control animals inoculated with the DY TME agent alone (p > 0.05). n.a., not applicable.To examine the question if prion interference can occur following a natural route of infection, hamsters were first inoculated per os with the DY TME agent and then superinfected per os with the HY TME agent at various time points post DY TME agent infection. Hamsters were per os inoculated by drying the inoculum on a food pellet and feeding this pellet to an individual animal as described previously.22 For the per os interference experiment, 105.7 i.c. LD50 of the DY TME agent or an equal volume of uninfected brain homogenate (100 µl of a 10% w/v brain homogenate) was inoculated 60, 90 or 120 days prior to per os superinfection of hamsters with 107.3 i.c. LD50 of the HY TME agent. A 60 or 90 day interval between DY TME agent infection and HY TME agent superinfection resulted in all of the animals developing clinical signs of HY TME with incubation periods that are similar to control hamsters inoculated with the HY TME agent alone (Fig. 1, lanes 11–16). The eight-day extension in the incubation period of HY TME in the 120 day interval co-infected group is consistent with a 1 log reduction in titer.21 This is the first report of prion interference by the per os route of infection, a likely route of prion infection in natural prion disease and provides further evidence that prion strain interference could occur in natural prion disease.23–25
Table 2
Clinical signs and incubation periods of hamsters per os inoculated with either the HY TME or DY TME agent, or per os co-infected with the DY TME agent 60, 90 or 120 days prior to superinfection of hamsters with the HY TME agent
Onset of clinical signs
First inoculation
Interval between inoculations
Second inoculation
Clinical signs
PrP-res migration
A/Ia
After 1st inoculation
After 2nd inoculation
Mock
120 days
HY TME
HY TME
21 kDa
5/5
n.a.
140 ± 5b
DY TME
60 days
HY TME
HY TME
21 kDa
5/5
195 ± 6
135 ± 6
DY TME
90 days
HY TME
HY TME
21 kDa
5/5
230 ± 5
140 ± 5
DY TME
120 days
HY TME
HY TME
21 kDa
5/5
269 ± 3
149 ± 3c
Open in a separate windowaNumber affected/number inoculated;bAverage days postinfection ± standard deviation;cIncubation period extended compared to control animals inoculated with the HY TME agent alone (p < 0.01); n.a., not applicable.The capacity of the DY TME agent to replicate modulates its ability to interfere with the HY TME agent. TME interference, following sciatic nerve inoculation, occurs in the lumbar spinal cord and DY PrPSc abundance in this structure correlates with the ability of the DY TME agent to interfere with the HY TME agent.12 Following extraneural routes of infection, DY TME agent replication and PrPSc deposition are not detected in spleen or lymph nodes, which is the major site of extraneural HY TME agent replication.11,21,26 The DY TME agent can interfere with the HY TME agent following intraperitoneal and per os infection, suggesting that the DY TME agent is replicating in other locations that are involved in HY TME agent neuroinvasion (11相似文献
The effects of analogs of diadenosine 5,5-p1,p4-tetraphosphate (Ap4A) were examined on the ADP-ribosylation reaction of histone Hl catalysed by purified bovine thymus poly(ADP-ribose)transferase. Among the compounds tested, Ap4A and ApCH2PPPA were shown to be the most efficient inhibitors of the enzyme. From kinetic studies of their action, it appears that Ap4A and ApCH2pppA might be mixed type inhibitors.Abbreviations ADP-ribose
adenosine diphosphate ribose
- ADPRT
poly-(ADP-ribose)transferase
- Ap4A
diadenosine 5,5-p1,p4-tertraphosphate
-
Ap4A
diadenosine 5,5-p1,p4(-1,N6-ethenyl-)tetra-phosphate
-
ApAA
diadenosine 5,5-p1,p4(-N6(-1,N6-)bisethenyl-)tetraphosphate
- ApCH2pppA
diadenosine 5,5-p1,p4(-p1,p2-methylene-)tetraphosphate
- AppCH2ppA
diadenosine 5,5-p1,p4(-p2,p3methylene-)tetraphosphate
- AppNHppA
diadenosine 5,5-p1,p4(-p2,p3-amino-)tetraphosphate
- AppCHBrppA
diadenosine 5,5-p1,p4(-p2,p3-bromine methyno-)tetraphosphate
- CpCH2ppCH2PC
dicytidine 5,5-p1,p4(-p1,p2-p3,p4-bismethylene-)tetraphosphate
- ApCH2ppCH2pA
diadenosine 5,5-p1,p4(-p1,p2-p3,p4-bismethylene-)tetraphosphate. 相似文献
The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrPSc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.PrPSc, a disease-specific marker for prion diseases and the likely infectious agent, is widely distributed within the central nervous system (CNS) and lymphoreticular tissues (LRS) in ovine scrapie, human variant Creutzfeldt-Jakob disease (vCJD), and cervine chronic wasting disease (CWD) during both clinical and preclinical stages (4, 11, 25). Furthermore, while the LRS distribution of PrPSc is much more restricted in bovine spongiform encephalopathy (BSE), sheep experimentally infected with BSE display a PrPSc tissue distribution more akin to that of ovine scrapie (11).For rodent-adapted scrapie and cervine CWD, the disease agent is detected in excreta when animals are in the clinical stages of disease, a process likely to contribute to environmental reservoirs of infectivity and lateral disease transmission (5, 13, 21). Within an experimental rodent model, it has also been demonstrated that the shedding of PrPSc and concomitant infectivity in feces occurs during preclinical scrapie (21).Evidence now also demonstrates that milk provides a vehicle for the transmission for prion diseases. Scrapie-free lambs fed milk from clinical scrapie-affected ewes propagate PrPSc within their LRS (8). Additionally, a recent study using a transgenic mouse bioassay demonstrated the secretion of infectivity in milk from preclinical animals where scrapie infectivity was found in milk months before the onset of clinical signs in animals with an ARQ/VRQ PrP genotype (10). The presence of scrapie infectivity within milk was irrespective of mammary gland pathology or PrPSc accumulation, and these animals were estimated to have considerable accumulation of immunohistochemically (IHC) detectable PrPSc within the LRS at the time of sampling.Here, we applied serial protein misfolding cyclic amplification (sPMCA) to the in vitro detection of PrPSc within sheep milk (Fig. (Fig.1)1) (Table (Table11).Open in a separate windowFIG. 1.sPMCA analysis of ovine milk samples. Milk was clarified and seeded into brain homogenate from sheep unexposed to the scrapie agent. Samples underwent sPMCA, and products were digested with proteinase K before analysis of 10 μl of each sample. PrP was detected with monoclonal antibodies SHA31 and P4; molecular mass markers are indicated (kDa). Milk was sampled from animals not exposed to the scrapie agent (U), those displaying clinical signs of scrapie (C), or those exposed to a scrapie-positive farm environment but not displaying clinical disease (S). NS, non-seeded PMCA brain substrate subjected to identical sPMCA conditions at the same time as positive samples were analyzed. Samples from the four nonexposed animals were analyzed 18 to 20 times each by sPMCA. Samples from clinically affected or clinically normal scrapie-exposed animals were analyzed in triplicate. For this triplicate analysis of each sample, the sPMCA round at which samples became positive is indicated under the appropriate lane. n, negative at round 12. Each sample was PrPSc negative until the stated round and thereafter was positive.
TABLE 1.
Timeline of exposure of animals to a scrapie-positive farm environment, sample collection, and scrapie status
Animala (PrP genotypeb)
Age at exposurec
Days postexposure at lactation
Days postlactation to clinical scrapied
Clinical statuse
PrPSc detection at postmortemf
PrPSc detection in milk (positive tests/total tests)g
1349/08 (VRQ/VRQ)
Not exposed
NAh,i
NA
Negative
Negative
0/20
K489 (VRQ/VRQ)
Not exposed
NAi
NA
Negative
NA (still alive)
0/18
0618/06 (VRQ/VRQ)
Not exposed
NAi
NA
Negative
Negative
0/20
1348/08 (VRQ/VRQ)
Not exposed
NAi
NA
Negative
Negative
0/20
0695/07 (VRQ/VRQ)
Birth
666-680
0
Positive
Positive
3/3
0334/07 (VRQ/VRQ)
Birth
661-666
0
Positive
Positive
3/3
0335/07 (VRQ/VRQ)
Birth
666-674
0
Positive
Positive
3/3
0350/07 (VRQ/VRQ)
Birth
663-676
0
Positive
Positive
3/3
0333/07 (VRQ/VRQ)
Birth
667-675
0
Positive
Positive
3/3
0142/07 (VRQ/VRQ)
Birth
665-673
0
Positive
Positive
3/3
0326/07 (VRQ/VRQ)
Birth
670-676
0
Positive
Positive
2/3
0199/07 (VRQ/VRQ)
Birth
664
0
Positive
Positive
2/3
0692/07 (ARQ/VRQ)
∼480 days
1,003
>450
Negative
Positive
2/3
0480/07 (ARQ/VRQ)
∼480 days
1,003
>355
Negative
Positive
3/3
0349/07 (ARQ/VRQ)
∼480 days
1,003
>348
Negative
Positive
3/3
0822/07 (ARQ/VRQ)
Birth
760
>564
Negative
Negative
2/3
2295 (AHQ/VRQ)
∼120 days
1,376
>621
Negative
Negative
3/3
3148 (ARR/VRQ)
Birth
1,288
>621
Negative
NA (still alive)
2/3
1514 (ARR/VRQ)
Unknown
597
>621
Negative
NA (still alive)
2/3
1518 (ARR/VRQ)
Unknown
597
>621
Negative
NA (still alive)
1/3
1244 (ARR/VRQ)
Birth
1,130
>621
Negative
NA (still alive)
3/3
Open in a separate windowaAll animal procedures were performed under Home Office (United Kingdom) and local ethical review committee approval and compliance with the Animal (Scientific Procedures) Act of 1986.bAmino acid residues at positions 136, 154, and 171 of the PRNP gene.cIntroduction into a scrapie-affected flock.dDays postlactation to postmortem or as of 12 December 2008 for animals that were still alive at the time this paper was written.eClinical disease usually included head tremors and pruritus with associated wool loss and nervousness. The indicated clinical status was applicable throughout lactation to either postmortem or as of 12 December 2008 for animals that were still alive at the time this paper was written.fPrPSc was analyzed by IHC, Western blot analysis, or enzyme-linked immunosorbent assay. All animals with a positive result contained PrPSc within both brain and lymphatic tissues.gsPMCA was used for PrPSc detection and the results are tallied within this column. Replica analysis of a single milk sample from each animal was carried out. For animals 0695/07, 0334/07, 0335/07, 0350/07, 0333/07, 0142/07, and 0326/07, multiple milk samples were collected during the lactation period indicated and multiple samples from an individual animal were pooled before analysis.hNA, not applicable.iNonexposed animals were 750 to 1,110 days old at lactation and where applicable were 1,200 to 1,650 days old at postmortem.PMCA was first described by Saborio and colleagues (20) and allows the amplification of minute quantities of PrPSc (18). In rodent scrapie models, this methodology has detected PrPSc in both blood (2, 18) and brain (22) material in the clinical and preclinical stages of disease as well as in urine excreted during clinical disease (14). This technique has recently been applied to the high-level amplification of PrPSc from natural hosts of prion diseases, including vCJD (7), CWD (9), and scrapie (23). Fresh ovine milk was obtained from individual sheep at least 7 days postpartum. Milk was collected from individual animals into sterile containers and stored on ice for shipping. Within 48 h of collection, milk samples were stored at −80°C. Colostrum was not analyzed. After thawing milk samples, samples from the same individual animal were pooled and EDTA, Nonidet P-40, and sodium deoxycholate were added to final concentrations of 50 mM, 0.5% (vol/vol), and 0.5% wt/vol, respectively. Samples (1 ml) were centrifuged for 10 min at 16,000 × g. After cooling on ice for 5 min, clarified milk supernatant was withdrawn from under the solidified fat layer.sPMCA was carried out as described by Thorne and Terry (23), who demonstrated that samples from a range of animals containing at least one VRQ PrP allele could be amplified by this technique. Clarified milk supernatant was diluted 1 in 10 into PMCA brain homogenate substrate (10% [wt/vol] brain homogenate from a VRQ/VRQ PrP genotype animal within 150 mM NaCl, 4 mM EDTA, pH 8.0, 1.0% [wt/vol] Triton X-100, and miniprotease inhibitor; Roche) to a final volume of 100 μl. Samples contained within sealed 0.2-ml PCR tubes were placed in a rack within an ultrasonicating water bath (model 3000; Misonix) that held the bottom of the tubes 0.4 cm above the sonicator horn. Water was added to the water bath up to the rack surface, immersing the sonicator horn. The water bath was held at 37°C, and sonications were performed for 40 s at 200 W, equivalent to 80% of the maximum power output of the machine. Sonications were repeated once every 30 min for 24 h (one PMCA round), after which the amplified samples were diluted 1 in 3 with PMCA substrate in a final volume of 100 μl and the sample was subjected to further rounds of PMCA. Twelve PMCA rounds were performed for each sample, a total of 576 sonications over 12 days. PMCA samples were digested with 50 μg/ml proteinase K for 1 h at 37°C before analysis of 10 μl of each sample by Western blotting using 12% (wt/vol) acrylamide NuPAGE precast Bis-Tris gels (as described in reference 15). All clinical scrapie-affected animals or those exposed to the scrapie agent were challenged by introduction into the Ripley flock (Veterinary Laboratories Agency, United Kingdom), where natural scrapie is endemic with a high incidence since 1996. Ryder and coworkers (17) reported that all animals with PrP genotypes VRQ/VRQ and ARQ/VRQ that were born into this flock developed scrapie, with incubation periods of 21 to 28 months and 28 to 39 months, respectively. When ARQ/VRQ animals were introduced into the flock at 6 to 26 months of age, 77% of the animals had subclinical scrapie 24 to 30 months later, as detected by IHC analysis of the LRS. Here, PrPSc was detected in the milk from clinically affected animals at a rate of 92% (24 analyses; triplicate analyses of samples from 8 animals) and from scrapie-exposed, clinically normal sheep at a rate of 78% (27 analyses; triplicate analyses of samples from 9 animals) (Fig. (Fig.1)1) (Table (Table1).1). All scrapie-exposed sheep, both clinically affected and clinically normal, tested positive for PrPSc in at least one sPMCA reaction. PrPSc was amplified from the milk of sheep with VRQ/VRQ, ARR/VRQ, ARQ/VRQ, and AHQ/VRQ PrP genotypes (Table (Table1).1). It required at least four to eight rounds of sPMCA to produce detectable PrPSc within a milk sample from each of the scrapie-exposed sheep (Fig. (Fig.1).1). Replica analysis of a pooled milk sample from each individual sheep occasionally demonstrated high variability in the round that samples became positive for PrPSc (Fig. (Fig.1);1); this result may indicate the presence of very low levels of PrPSc (19) and/or heterogeneity within milk samples. Analyses of ovine milk from a New Zealand-derived scrapie-free flock kept under strict biosecurity conditions (ADAS, United Kingdom) did not amplify PrPSc within 12 rounds of sPMCA (78 analyses; up to 20 replica analyses of samples from 4 animals). For each of the sPMCA analyses, both positive and negative samples were analyzed concurrently within the same run on the same sonicator. These data demonstrate that PrPSc amplified from samples from scrapie-exposed animals is not due to spontaneous PrPSc formation or cross-contamination between samples within the sPMCA procedure. It is of note that prions were shed within milk from clinically normal, scrapie-exposed animals with multiple PrP genotypes. The ARQ/VRQ genotype is indicative of a high level of disease penetrance and widespread preclinical PrPSc accumulation within the LRS system, whereas AHQ/VRQ and ARR/VRQ genotype animals typically have much lower disease penetrance (24) and LRS involvement (11). This indicates the secretion of prions within milk regardless of high-level PrPSc accumulation within the LRS and also the very likely detection of subclinical as well as preclinical disease in some of these animals.No clinical scrapie-affected animals displayed clinical mastitis, and PrPSc was not detected within mammary gland tissue from five sheep with clinical scrapie (Fig. (Fig.22 and data not shown). This is in agreement with the study by Lacroux et al. (10), indicating that while the accumulation of PrPSc within mammary gland tissue can occur, it is not a prerequisite for its deposition within milk. Here, postmortem detection of PrPSc was carried out by routine diagnosis using IHC and Western blot analysis of the obex. Exceptions were animals 1349/08 and 1348/08, where obex tissue was analyzed by Bio-Rad TeSeE enzyme-linked immunosorbent assay (Table (Table1).1). Postmortem IHC examination of palatine tonsil, ileal Peyer''s patches, medial retropharyngeal lymph node, and mesenteric lymph node tissue was also carried out. Scrapie-exposed animals were shown to secrete PrPSc within their milk irrespective of whether they could be confirmed as scrapie positive by postmortem immunoassay detection of PrPSc within the CNS and LRS (Table (Table1).1). This discrepancy in PrPSc detection may well reflect the greater sensitivity of sPMCA compared to immunoassay detection of PrPSc; these results also indicate that PrPSc is secreted during the early stages of disease progression. Scrapie-exposed animals had PrPSc detected within their milk at least 20 months prior to possible clinical onset of disease, and this was not apparently influenced by the PrP genotype.Open in a separate windowFIG. 2.Detection of protease-resistant PrPSc within CNS and mammary gland tissues of animals displaying clinical scrapie. Tissues were prepared as 10% or 40% (wt/vol) homogenates for spinal cord and mammary gland tissue, respectively, as described previously elsewhere (15). Native or proteinase K-digested homogenates (25 μg/ml; 1 h at 37°C) were analyzed as indicated. Protease-resistant PrPSc was readily detectable within spinal cord tissue (SC; lanes 1 to 2) but was not detectable within mammary gland samples (MG; lanes 3 to 6). Either 0.33 mg (0350/07) or 0.165 mg (0326/07 and 0344/07) of spinal cord tissue and 1.32 mg (lanes 3 and 4) and 6 mg (lane 5) of mammary gland tissue was analyzed per lane. Protease-resistant PrPSc was still undetectable from 20 mg of mammary gland tissue following precipitation with sodium phosphotungstic acid (25) prior to analysis (lane 6). PrPSc within scrapie-positive brain tissue (63 μg) was readily detected by this method after spiking into 20 mg of mammary gland homogenate (B, lane 7). Full-length and fragmented protease-sensitive PrPC was readily detected within mammary gland tissue (lane 3). Animal numbers are indicated. PrP was detected with monoclonal antibody SHA31; molecular mass markers are indicated (kDa).These data clearly demonstrate that the secretion of PrPSc within milk occurs in natural scrapie. There are several routes through which the prion protein could be secreted into milk. Evidence suggests that within ovine mammary gland tissue, PrPC is actively produced within epithelial cells, and its secretion is most likely by exocytosis and the apocrine secretion of fat globules (3). It is unknown whether PrPSc is produced within epithelial cells and secreted into milk through similar mechanisms. Alternative mechanisms are through vesicular transcytosis or paracellular transport of PrPSc from the blood. It is established that prion-infected animals harbor infectivity and PrPSc within the blood during preclinical disease (6, 18) and that blood components are secreted within milk, including cell types known to colocalize with PrPSc within ovine mammary glands (12).Results indicate the potential transmission of scrapie in the milk of infected sheep for a prolonged period prior to clinical onset. As well as ewe-to-lamb disease transmission, this process is also likely to contribute to lateral transmission, as lambs fed milk from clinically infected ewes were the source for the transmission of scrapie between lambs within the first few months after birth (8). It is unknown whether other prion diseases result in the secretion of prions within milk. CWD, vCJD, and experimental ovine BSE share similarities with scrapie in the tissue distribution of infectivity, and it seems plausible that an analogous secretion mechanism may occur. Given the extended preclinical stages and the purported importance of subclinical states for these diseases (16), such an outcome would have significant implications for the transmission of prion diseases from apparently healthy animals and humans.With regard to ovine milk and milk products, scrapie is not transmissible to humans, and to date there is no evidence of the natural occurrence of ovine BSE. As such, the reported findings do not indicate the likely introduction of zoonotic prions from sheep into the human food chain. Nevertheless, the presented data do indicate caution in the risk assessment associated with such foods. Also, it is unknown if analogous shedding of prions into milk occurs with bovine BSE; evidence from previous epidemiological and bioassay studies would suggest that such a scenario seems unlikely to cause clinical disease (1, 26). However, the present report demonstrates that prions are secreted within the milk of sheep with PrP genotypes not typically associated with LRS accumulation of PrPSc and that prions were secreted from animals devoid of IHC-detectable PrPSc within their LRS. Such PrPSc tissue distribution is similar to bovine BSE, and given the importance of bovine milk in the human diet, the potential presence of low levels of prions within bovine milk warrants further investigation.Finally, analyzing milk samples by sPMCA offers a methodology with a clear potential for the identification of clinically sick animals and those with preclinical/subclinical scrapie. Such a noninvasive live-animal assay has the potential to contribute to the epidemiological study, management, and control of prion diseases within farmed animals. 相似文献
Cannabinoids were found to augment phospholipase activities and modify lipid levels of mouse brain synaptosomes, myelin and mitochondria. Delta-1-tetrahydrocannabinol (1-THC) and several of its metabolites induced a dose-dependent (0.32–16 M) stimulation of phospholipase A2 (PLA2) activity resulting in the increased release of free arachidonic acid from exogenous [1-14C]phosphatidylcholine (PC). The potencies of the cannabinoids in modulating PLA2 activity were approximately of the order: 7-OH-1-THC > 1-THC > 7-oxo-1-THC > 1-THC-7oic acid = 6 OH-1-THC 6-OH-1-THC. The hydrolysis of phosphatidylinositol (PI) by synaptosomal phospholipase C (PLC) was enhanced significantly by 1-THC and promoted diacylglyceride levels by greater than 100 percent compared to control values. In contrast, arachidonate was the major product resulting from phospholipase activities of a 20,000g pellet. Synaptosomal diacylglyceride lipase activity was inhibited by 1-THC. [1-14C]Arachidonic acid was readily incorporated into subcellular membrane phospholipids and after exposure to cannabinoids led to diminished phosphoglyceride levels and concomitant increases in released neutral lipid products. These data suggest that cannabinoids control phospholipid turnover and metabolism in mouse brain preparations by the activation of phospholipases and, through this mechanism, may exert some of their effects. 相似文献
The degradation process of acephate in aqueous solution with OH and produced by 60Co-γ irradiation and electron pulse radiolysis was studied in the present paper. In the aqueous solution, acephate reacted with and transformed to transient species which can absorb weakly in the wavelength range of 300–400?nm and decay very fast. According to the decay of hydrated electron, the reaction rate constant of and acephate is (3.51?±?0.076)?×?109?dm3·mol?1·s?1. The transient species produced in the reaction of OH and acephate do not distinctly absorb the light in the wavelength range of 300–700?nm, so the decay and kinetics of the transient species cannot determinedirectly. The competing reaction of KSCN oracephate with OH were studied to obtain the reaction rate constant of OH and acephate, which is (9.1?±?0.11)?×?108?dm3·mol?1·s?1. Although acetylamide and inorganic ions were determined in the products of the reaction of acephate with OH or , the concentration of inorganic ions in the products of the reaction of acephate with OH is higher than that in the product of the reaction of acephate with . Moreover, there were sulfide in the products of the reaction of acephatewith . The degradation pathways of acephate by OH and were also proposed based on the products from GC-MS. 相似文献
Stable (13C and 15N) and radio- (14C, 137Cs and 210Pb) isotopes were determined for termites that
have been sampled from a dry evergreen forest in Thailand. A wood-feeding termite, Microcerotermes crassus, was separated from soil-feeders: Termes propinquus, Termes comis and Dicuspiditermes makhamensis by 13C and 15N values. The Termes group in Thailand had less diverse values in 13C and 15N than those in Australia, where the feeding habits of the Termes group are more diverse. Other soil-feeding termites produced similar 13C values, but a larger range in 15N values. 14C-percent modern carbon (pMC) values suggest that the soil-feeding termites used younger carbon than the wood-feeding termites, and this was consistent with the termites from Cameroon, central Africa. Values of 13C and 14C-pMC indicate that surface soil was used by a soil-feeding termite, D. makhamensis, in making the nest mounds, and deeper soil (10–30 cm) by a fungus-growing termite, Macrotermes carbonarius. 210Pb and 137Cs were scarcely incorporated into the termites, although 214Pb was recovered from the workers. The results suggest that stable- and radioisotopes are useful in the study of detritivorous animals, organic matter decomposition and ecosystem engineering.Takuya Abe - deceased. 相似文献
The photoreceptors for chloroplast photorelocation movement have been known, but the signal(s) raised by photoreceptors remains unknown. To know the properties of the signal(s) for chloroplast accumulation movement, we examined the speed of signal transferred from light-irradiated area to chloroplasts in gametophytes of Adiantum capillus-veneris. When dark-adapted gametophyte cells were irradiated with a microbeam of various light intensities of red or blue light for 1 min or continuously, the chloroplasts started to move towards the irradiated area. The speed of signal transfer was calculated from the relationship between the timing of start moving and the distance of chloroplasts from the microbeam and was found to be constant at any light conditions. In prothallial cells, the speed was about 1.0 µm min−1 and in protonemal cells about 0.7 µm min−1 towards base and about 2.3 µm min−1 towards the apex. We confirmed the speed of signal transfer in Arabidopsis thaliana mesophyll cells under continuous irradiation of blue light, as was about 0.8 µm min−1. Possible candidates of the signal are discussed depending on the speed of signal transfer.Key words: Adiantum capillus-veneris, Arabidopsis thaliana, blue light, chloroplast movement, microbeam, red light, signalOrganelle movement is essential for plant growth and development and tightly regulated by environmental conditions.1 It is well known that light regulates chloroplast movement in various plant species. Chloroplast movement can be separated into three categories, (1) photoperception by photoreceptors, (2) signal transduction from photoreceptor to chloroplasts and (3) movement of chloroplasts and has been analyzed from a physiological point of view.2 We recently identified the photoreceptors in Arabidopsis thaliana, fern Adiantum capillus-veneris, and moss Physcomitrella patens. In A. thaliana, phototropin 2 (phot2) mediates the avoidance movement,3,4 whereas both phototropin 1 (phot1) and phot2 mediate the accumulation response.5 A chimeric photoreceptor neochrome 1 (neo1)6 was identified as a red/far-red and blue light receptor that mediates red as well as blue light-induced chloroplast movement in A. capillusveneris.7 Interestingly, neo1 mediated red and blue light-induced nuclear movement and negative phototropic response of A. capillus-veneris rhizoid cells.8,9 On the mechanism of chloroplast movement, we also found a novel structure of actin filaments that appeared between chloroplast and the plasma membrane at the front side of moving chloroplast.10 Recent studies using the technique of microbeam irradiation have revealed that chloroplasts do not have a polarity for light-induced accumulation movement and can move freely in any direction both in A. capillus-veneris prothallial cells and in A. thaliana mesophyll cells.11 However, the signal that may be released from photoreceptors and transferred to chloroplasts remains unknown.To understand the properties of the signal for the chloroplast accumulation response, we examined the speed of signal transfer in dark-adapted A. capillus-veneris gametophyte cells and A. thaliana mesophyll cells by partial cell irradiation with a red and/or blue microbeam of various light intensities for 1 min and the following continuous irradiation, respectively.12As shown in Figure 1, the relation between the distance of chloroplasts from the microbeam and the timing when each chloroplast started moving toward the microbeam irradiated area (shown as black dots in Fig. 1) was obtained and plotted. The lag time between the onset of microbeam irradiation and the timing of start moving of chloroplasts is the time period needed for a signal to reach each chloroplast. To obtain more accurate data many chloroplasts at various positions were used. The slope of the approximate line indicates the average speed of the signal transfer. Shown with a protonemal cell at the left side of this figure is an instance where the speed of signal transfer from basal-to-apical (acropetal) direction is obtained.Open in a separate windowFigure 1How to calculate the speed of signal transfer in the basal cell of two-celled protonema of Adiantum capillus-veneris. The relationship between the distance of chloroplast position from the edge of the microbeam to the center of each chloroplast as shown in left side of figure and the timing of chloroplast movement initiated shown as the black dots was obtained. Inclination of the approximate lines connecting dots indicates the speeds of the signal transfer.In protonemal cells, which are tip-growing linear cells, the average speed of signal transfer was about 2.3 µm min−1 from basal-to-apical (acropetal) and about 0.7 µm min−1 from apical-to-basal (basipetal) directions. These values were almost constant irrespective of light intensity, wavelength, irradiation period, and the region of the cell irradiated.12 The difference of speed between basipetal and acropetal directions may be depending on cell polarity. The signal transfer in prothallial cells of A. capillus-veneris and mesophyll cells of A. thaliana was about 1.0 µm min−1 to any direction, probably because they may not have a polarity comparing to protonemal cells or have a weak polarity if any. Thus, the speed of signal transfer must be conserved in most land plants,12 if not influenced by strong polarity.
Open in a separate windowThe speeds of signal transfer under different light intensities and wave length in Adiantum capillus-veneris gametophyte cells and Arabidopsis thaliana mesophyll cells are summarized. When dark-adapted cells were irradiated with various light intensities (red light: 10, 1, 0.1 W m−2) of a microbeam of red or blue light for 1 min or continuously, the chloroplasts moved towards the irradiated area. The speed of signal transfer was measured from the relationship between the timing of onset of moving and the distance of chloroplalsts from the microbeam irradiated area.Calcium ions have been proposed as one of the candidates of the signal. Calcium is reported to be necessary for chloroplast movement in some plants.13,14 Chloroplast movement under polarized light could not be induced in the existence of EGTA in protonemal cells of A. capillus-veneris, although chloroplasts show slight movement in random direction.13 In Lemna trisulca, chloroplast movement correlates with an increase of cytoplasmic calcium levels and is inhibited by antagonists of calcium homeostasis.14 The speed of intracellular transfer of calcium ions in plant cells was measured only in moss Physcomitrella patens by microinjection of a calcium indicator into protonemal cells.15 The speed of calcium waves in the cytoplasm of protonemal cell was about 3.4 µm sec−1. The speed of substance transfer as signals is not known in plant cells except for the above instance, as far as we know, but in animal cells various experimental data has been accumulated.16–21The transfer speed of calcium waves visualizing cytoplasmic free calcium by microinjection of aequorin was about 8 µm sec−1 in Xenopus eggs.16 Calcium ion expands as a spherical wave and the wave speed in plane is 50 µm sec−1 in rat cardiac myocytes when measured by loading a membrane-permeable indicator of calcium into the cell. The maximum velocity was 112 µm sec−1.17 Calcium waves could also be observed in the SR-free single isolated rabbit cardiac myofibrils with a propagation velocity of 15.5 µm sec−1.18 The propagation velocity of the calcium wave was about 65–100 µm sec−1 by calciuminduced calcium release (CICR) in pig heart muscle cells.19–21 Comparing these values to our data in A. capillus-veneris, the speed of signal transfer in chloroplast movement in fern gametophytes was 100–200 times slower than those measured for calcium ion transfers in animal cells, suggesting that the calcium might not be the signal involved in chloroplast movement.Intracellular transport is depended on the cytoskeleton systems in many cases. So the speed of movement of the cytoskeleton itself has been examined. When motor-proteins (such as 22s dynein, 14s dynein, kinesin) were anchored on a slide glass microtubules overlaid moved with a speed of about 4.52, 4.29, 0.422 µm sec−1, respectively. In similar ways, actin filaments placed over myosin-coated glass moved at about 5.21 µm sec-1.22 On the other hand, the motor domain of the Centromere Binding Factor (CBF) protein complex moves at 4.04 µm min−1 on microtubules.23 In A. capillus-veneris protonemal cells, the speed of cytoplasmic streaming depending on the actomyosin system was calculated from the speed of oil drop movement.24 The speed was dependent upon the position of long protonemal cells and was about 2 µm min−1 in the apical region and gradually increased to 10 µm min−1 in the basal region. In comparison to the data cited here, the speed of signal transfer involved in chloroplast accumulation was 30–120 times slower than the speed of the actomyosin system or the microtubule-kinesin/dynein system, but it is similar to the moving speed of a protein complex on a microtubule23 and oil droplets in a protonemal cell.24Polymerization rates of cytoskeletal proteins have been measured using in vitro systems. For instance, the plus end of microtubules from bovine brains grew at 1.04–1.88 µm min−1.25,26 Polymerization rate of actin filaments from rabbit muscle was about 0.13–0.49 µm min−1 and depended on the G-actin concentration.27 Live BHK21 fibroblasts, mouse melanoma cells and Dictyostelium amoebae expressing GFP-actin fusion proteins move on glass by using three-dimensional F-actin bands. These structures propagate throughout the cytoplasm at rates ranging between 2–5 µm min−1 in each cell type and produce lamellipodia or pseudopodia at the cell boundary.28 The extending speed of these cytoskeletons is roughly equal to the speed of signal transfer for the chloroplast accumulation response. We therefore aim to measure the speed of extension of these filaments when a method of gene transformation has been established for A. capillus-veneris. 相似文献
Four point-of-use disinfection technologies for treating sewage-contaminated well water were compared. Three systems, based on flocculant-disinfectant packets and N-halamine chlorine and bromine contact disinfectants, provided a range of 4.0 to >6.6 log10 reductions (LR) of naturally occurring fecal indicator and heterotrophic bacteria and a range of 0.9 to >1.9 LR of coliphage.Disasters and flooding can overwhelm sanitation infrastructure, leading to sewage contamination of potable waters. This may be routine during the wet season in many parts of the world and spreads numerous waterborne diseases (21). Point-of-use (POU) water treatment has reduced the incidence of diarrheal disease when used for household drinking water (3, 4, 6, 13) and is now being promoted for disaster relief. While POU systems have recently been reviewed (14), to our knowledge there has been no direct, experimental comparison for treating actual sewage-contaminated waters. In this study, the efficacies of four POU disinfection systems (based on sodium dichloroisocyanurate [NaDCC] tablets, a flocculent-disinfectant powder, and chlorine and bromine contact disinfectant cartridges) in reducing the concentrations of six microbial indicators in well water contaminated with raw sewage were compared.The NaDCC tablets (67 mg; Aquatabs; Medentech, Wexford, Ireland), used for disinfection in low-turbidity water, have shown preliminary efficacy for routine household drinking water treatment (3, 4). The flocculant-disinfectant packet (4 g; PUR; Procter & Gamble Co., Cincinnati, OH) includes Fe2(SO4)3, bentonite, Na2CO3, chitosan, polyacrylamide, KMnO4, and Ca(OCl)2 (13). It achieved >7.3 log10 reductions (LR) of 24 bacteria species; >4.6 LR of poliovirus and rotavirus in EPA no. 2 test water (turbidity, >30 nephelometric turbidity units [NTU]) (15); and reduced diarrheal illness in Guatemala, Liberia, Kenya, and Pakistan (6, 7, 11, 13).HaloPure canisters (Eureka Forbes, Mumbai, India) contain N-halamine polymer disinfectant beads, poly[1,2-dichloro-5-methyl-5-(4′-vinylphenyl)hydrantoin] for chlorine canisters, and poly[1,2-dibromo-5-methyl-5-(4′-vinylphenyl)hydrantoin] for bromine canisters. Seeded laboratory trials achieved >6.8 LR for Escherichia coli and Staphylococcus aureus as water was passed through the canisters (2). The Cl-contact (producing residuals ranging from 0 to 0.6 mg/liter) and Br-contact (with residuals of 0.68 to 1.8 mg/liter) disinfectants achieved 2.9 LR and 5.0 LR of the bacteriophage MS2, respectively, and 27.5% and 88.5% reductions of the algal toxin microcystin, respectively (5).Sewage-contaminated water was prepared by mixing 9 liters of potable, nonchlorinated well water (pH 7.8; turbidity, 0.33 NTU; Williamston, MI) with 1 liter of raw sewage (City of East Lansing Wastewater Treatment Plant, MI) with an average pH of 6.6 ± 0.1, a biochemical oxygen demand of 144 ± 36 mg/liter, a concentration of total suspended solids of 146 ± 31 mg/liter, and a turbidity of 132 ± 12 NTU. Three disinfection trials were conducted at room temperature for each POU system on three different days to allow for variance in sewage strength. The turbidities of 1:10 dilutions of raw sewage averaged 7.5 ± 2.0 NTU. Table Table11 lists the indicator microorganism concentrations in the influent and effluent for each system.
TABLE 1.
Concentrations of influent and 30-min-effluent microorganisms for POU disinfectant systems treating sewage-contaminated water
Microorganism group
Geometric mean concn (range) [% of samples below detection limit]a
NaDCC
Flocculant-disinfectant
Cl-contact
Br-contact
Influent
Effluent at 30 min
Influent
Effluent at 30 min
Influent
Effluent at 30 min
Influent
Effluent at 30 min
Total coliforms
2.7 × 104 (6.7 × 103 to 7.6 × 104)
4.3 (4.0 × 10−2 to 1.6 × 102)
1.7 × 104 (1.2 × 104 to 2.7 × 104)
4.0 × 10−2 (<1.0 × 10−2 to 2.4 × 10−1) [33]
2.9 × 104 (2.3 × 104 to 4.0 × 104)
<1.0 × 10−2 [100]
4.5 × 104 (1.9 × 104 to 7.2 × 104)
1.1 × 10−2 (<1.0 × 10−2 to 1.3 × 10−2) [66]
Heterotrophic plate counts
8.7 × 104 (2.7 × 104 to 1.8 × 105)
6.4 × 101 (2.1 × 101 to 4.5 × 102)
8.9 × 104 (2.9 × 104 to 4.3 × 105)
8.5 (4.7 to 2.7 × 101)
6.6 × 104 (3.5 × 104 to 1.1 × 105)
3.9 (3.5 to 4.2)
8.3 × 104 (2.4 × 104 to 2.0 × 105)
4.6 (2.2 to 7.7)
E. coli
3.3 × 103 (7.7 × 102 to 1.1 × 104)
1.8 × 101 (9.0 × 10−1 to 5.3 × 102)
6.7 × 103 (2.3 × 103 to 4.3 × 104)
1.1 × 10−2 (<1.0 × 10−2 to 1.3 × 10−2) [66]
4.7 × 103 (2.3 × 103 to 1.1 × 104)
<1.0 × 10−2 [100]
1.5 × 104 (6.3 × 103 to 4.6 × 104)
<1.0 × 10−2 [100]
Enterococci
8.8 × 102 (5.7 × 102 to 1.3 × 103)
2.3 (<1.0 × 10−2 to 4.9 × 101) [33]
6.3 × 102 (5.0 × 102 to 8.7 × 102)
<1.0 × 10−2 [100]
9.9 × 102 (5.3 × 102 to 1.7 × 103)
<1.0 × 10−2 [100]
1.3 × 103 (7.3 × 102 to 2.3 × 103)
<1.0 × 10−2 [100]
Clostridia
1.6 × 102 (6.0 × 101 to 3.0 × 102)
6.4 (6.7 × 10−1 to 7.7 × 101)
2.0 × 102 (7.0 × 101 to 6.0 × 102)
7.9 × 10−1 (4.5 × 10−1 to 1.4)
3.4 × 101 (2.0 × 101 to 6.3 × 101)
2.4 × 10−2 (<1.0 × 10−2 to 6.0 × 10−2) [33]
4.4 × 101 (2.7 × 101 to 9.3 × 101)
7.4 × 10−2 (<1.0 × 10−2 to 3.6 × 10−1) [33]
Coliphage
1.5 × 102 (1.2 × 102 to 2.2 × 102)
3.1 × 101 (<1.0 to 1.8 × 102) [33]
1.4 × 102 (1.3 × 102 to 1.4 × 102)
1.9 × 101 (<1.0 to 1.1 × 102) [33]
9.4 × 101 (4.3 × 101 to 1.6 × 102)
7.3 (1.3 to 4.7 × 101)
7.7 × 101 (4.0 × 101 to 1.2 × 102)
<1.0 [100]
Open in a separate windowaValues shown are numbers of CFU/ml except those for coliphage, which are numbers of PFU/ml. The percentage of samples below the detection limit (n = 3 for all systems) is 0% if not shown.All systems were used in accordance with the manufacturer''s directions for 10 liters of water. For NaDCC trials, one tablet was added and allowed 30 min of contact time (total dose of 3.2 mg/liter of hypochlorite; in deionized water, one tablet produced 2.1 mg/liter free Cl residual). For flocculant-disinfectant trials, one packet was added, stirred vigorously for 5 min, strained through cheesecloth after 10 min, and allowed 20 min of further contact time. The amount of hypochlorite included in one packet was not indicated, but one packet provided 1.5 mg/liter free Cl residual in 10 liters of deionized water. Samples were taken at 1, 3, 5, 10, 15, and 30 min for both systems.For the Cl-contact and Br-contact trials, disinfectant cartridges were installed in AquaSure housings consisting of an upper reservoir for influent, which flows by gravity through the disinfectant cartridge to a lower reservoir with a tap for dispensing (Fig. (Fig.1).1). The housings usually include cloth and activated charcoal prefilters, but these were removed in order to directly evaluate the disinfectant. With the tap open, 10 liters of influent was added and samples were collected at first flow (6 to 12 min) and after 15 and 30 min of flow. A single chlorine canister was used for all trials; the bromine canister was replaced for the third trial because the original clogged.Open in a separate windowFIG. 1.Flow schematic for contact disinfectant cartridges. Arrows indicate the directions of water flow from the upper reservoir (U), through the halogen (chlorine or bromine) disinfectant cartridge (H) containing packed N-halamine beads (N), to the lower reservoir (L) and out through the open tap.Microbial indicators in the influent and effluent (collection tubes contained sodium thiosulfate) in triplicate were quantified as numbers of CFU/ml by using mENDO agar for total coliforms (9), mHPC agar for heterotrophic plate counts (8), mTEC medium for E. coli (19), mEI agar for the genus Enterococcus (18), and mCP agar for the genus Clostridium (1) (Becton, Dickinson and Co., Franklin Lakes, NJ). Coliphage (PFU/ml) were measured with a double agar overlay assay, EPA method 1601 (17). Residuals (mg/liter) were measured using a Hach chlorine (free and total) test kit, model CN66 (Hach Co., Loveland, CO) (used for bromine in accordance with Hach method 8016 [10], with the instrument reading multiplied by 2.25 [the ratio of the atomic weights of bromine and chlorine], as advised by Hach Co. technical support).Comparison of water quality levels was done at 30 minutes. LR were calculated, with zeros replaced with the detection limits (Fig. (Fig.2).2). All POU systems reduced microbial concentrations below the detection limit in some trials (Table (Table1),1), making the calculated reductions the lower bound for those trials.Open in a separate windowFIG. 2.Average LR of naturally occurring microorganisms at 30 min for sewage-contaminated well water (1:10 dilution of raw sewage in well water) with the use of four POU disinfection systems (error bars represent 1 standard error). * indicates that effluent was below the limit of detection for all samples. Limit of detection was substituted to calculate LR and actual reductions may be greater than shown.Average LR for each POU system were compared using two-way analysis of variance with post hoc least-significant-difference (LSD) tests, performed with SPSS 11.0.1 (SPSS, Inc.). LR at 30 min differed significantly between systems (analysis of variance; F3,5 = 20.6; P < 0.001). There was no significant difference between the LR achieved by flocculant-disinfectant and contact disinfectants (LSD; mean difference, 0.2 to 0.5 LR; P > 0.05), while the NaDCC tablets induced significantly lower reductions (LSD; mean difference, 1.5 to 2.0 LR; P < 0.001).There was detectable residual free chlorine after 30 min for one NaDCC trial (0.4 mg/liter) and two flocculant-disinfectant trials (0.1 and 0.4 mg/liter). No contact disinfectant trial produced a measurable residual.No system in this study reliably produced residuals for safe storage after POU treatment or ideal virus reduction. Except for the NaDCC system, the POU systems achieved approximately 5.5 LR for E. coli and coliforms, 4.5 LR for enterococci, 4.0 LR for heterotrophs, 2.5 LR for clostridia, and 1.0 LR for coliphage. Coliphage was reduced below detection limits in all trials with Br-contact, similar to what was found in previous research (5). Bromine disinfection has proved safe and effective for large-scale maritime applications, like U.S. Navy vessels (20), and appears promising for household treatment. Further assessment of the Br-contact system is warranted, as is field comparison of POU systems in disaster relief. 相似文献
Summary The occurrence of the AT chain (i.e. A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS1 patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were AT heterozygotes. The chromosomes with the AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to G; Hind III in G and A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for AT negative chromosomes.2 This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA 相似文献
We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca2+ influx blocking agents LaCl3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl3 and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca2+ influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl3 and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca2+]cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth.Key words: calcium influx, phosphoprotein, pollen tube growth, RIC protein, rop GTPaseRop (Rho GTPase of plants) was newly reported as a master regulator for plant signaling.1,2 It participates in concerted actions of many signaling pathways that influence growth and development, and the adaptation of plants to various environmental situations.3–5 In contrast to be negative regulators in ABA signaling,6 Rops might work as positive regulators in auxin signaling pathways.7,8 Recently, we have reported unique desiccation-associated ABA signaling in which the LLP-Rop1 gene is not only negatively regulated by desiccation but also positively regulated by developmental cues independent of ABA during pollen maturation.9 Although LLP-Rop1 and its target, LLP12-2, accumulate in abundance in the matured and dried pollen upon dehydration, the activity of LLP-Rop1 and LLP12-2 is likely restricted at the stage of pollen maturation.9 As pollen germinates, ABA content decreases its level in the growing tube and thus, the activity of Rop is less restricted than that in the dried pollen and subsequently Rops become powerful regulators playing crucial roles during pollen tube growth.Pollen germination and tube growth are a continuous and highly polarized process characteristic of tip growth. As soon as pollen hydrates and germinates, a tip-focused cytoplasmic Ca2+ gradient is established and sustained while a pollen tube grows forward.10,11 The Ca2+-permeable channels that modulate [Ca2+]cyt influx in germinating pollen grains have been identified in Arabidopsis,12,13 lily14 and pear.15 When a pollen tube grows, Rop-interactive Cdc42/Rac-interactive binding (CRIB) motif-containing proteins (RICs) play an important role as Rop GTPase targets and control a variety of Rop-dependent signaling pathways.16 RICs contain a CRIB motif required for their specific interaction with GTP-bound Rop. They are grouped into five classes that share little sequence similarity outside of the Rop-interactive domain.16 Different RICs expressed in various reproductive and vegetative parts of the Arabidopsis plant may act as Rop targets to control different Rop-dependent pathways in pollen tubes and in other organ development. For instances, RIC4 has been demonstrated to promote F-actin assembly, whereas RIC3 activates Ca2+ signaling by affecting [Ca2+]cyt influx that subsequently results in F-actin disassembly in pollen tube growth.17 The two RICs, both activated by AtRop1, counteract each other to control the actin dynamics and polar pollen tube growth.17We have demonstrated that LLP12-2, a RIC protein, interacts with active LLP-Rop1 in vivo.9 To examine the function of LLP12-2 in the growing tubes, the purified LLP12-2 PCR product digested with XbaI and SacI was cloned into the corresponding sites of Zm13::GFP construct to generate the Zm13::GFP-LLP12-2 construct. The transient expression of GFP-LLP12-2 in pollen using particle bombardment was investigated. Pollen germination and tube length were measured after particle bombardment and subsequent in vitro germination. With the treatment of Ca2+ influx blocking agents LaCl3 and EGTA, pollen expressing GFP alone significantly decreased germination and tube elongation, suggesting that a decrease in [Ca2+]cyt influx may cause the inhibition of pollen germination and tube growth (Fig. 1 and 17Open in a separate windowFigure 1LLP12-2 inhibits pollen germination by regulating the calcium influx channels. Germination percentages were determined 9 h after particle bombardment and subsequent in vitro germination. Equal amounts of GFP and LLP12-2 DNA (7.5 µg) were transiently expressed in lily pollen after which pollen was treated without (control) or with either LaCl3 (1 µM), EGTA (0.5 mM), LatB (0.05 nM), okadaic acid (5 nM) or staurosporine (1 µM) during germination.
Table 1
Effects of LaCl3, EGTA, LatB, okadaic acid or staurosporine on the length of pollen tube expressing LLP12-2
Pollen tube length (µm)
Control
LaCl3
EGTA
LatB
Okadaic acid
Staurosporine
GFP
>1,500
1,286 ± 26
1,262 ± 23
1,248 ± 32
1,265 ± 36
1,254 ± 31
LLP12-2
1,028 ± 22
1,305 ± 34
1,309 ± 31
1,021 ± 24
1,089 ± 35
1,186 ± 23
Open in a separate windowPollen tube length was measured 9 h after particle bombardment. Data are mean ± SD (µm) of three individual experiments (n = 10, per experiment).The inhibition of germination and tube growth was further enhanced in the pollen overexpressing GFP-LLP12-2 when compared with the pollen expressing GFP only (Fig. 1 and Fig. 1 and Fig. 1 and Fig. 1 and Fig. 1 and Figure 2. Any perturbation of [Ca2+]cyt influx in the pollen would decrease germination and tube growth. The function of LLP12-2 mimics that of RIC3, Group III of Arabidopsis RICs family. It has been reported that RIC3 activates Ca2+ signaling, which leads to F-actin disassembly, whereas RIC4 promotes F-actin assembly.17 Alike RIC3, overexpression of LLP12-2 causes an excess amount of tip-localized calcium in the cytoplasm of the tube and subsequently results in the inhibition of germination and tube growth. It should be noted that the LLP12-2-induced inhibition of germination and tube growth only partially rescued with the treatment of LaCl3 or EGTA, implying that factors other than calcium involves in the modulation of pollen germination and tube growth.18–20Open in a separate windowFigure 2Schematic diagram of the LLP-Rop1 signaling during pollen germination and tube growth. During germination and tube growth, LLP-Rop1 is activated at the tip and activates LLP12-2, which affects calcium influx in the cytoplasm that in turn promotes germination and tube elongation. In addition, staurosporine-sensitive protein kinases are involved in the LLP12-2-induced inhibition of pollen tube elongation.Protein kinases such as calcium-dependent protein kinase (CDPK) have been reported to involve in the regulation of pollen germination and tube growth.21,22 Studies have shown that CDPK comprises a kinase domain and a calmodulin-like domain in a single protein. Thus, it acts not only as a Ca2+ sensor but also as an effector affecting growth polarity, elevated cytosolic Ca2+, and plant cytoskeleton during pollen germination and tube growth.21,23 Aside from CDPKs, calcineurin B-like proteins (CBLs), a new family Ca2+ sensor, interact specifically with CBL-interacting protein kinases.24 These putative Ca2+ sensors are responsible for the regulation of calcium-dependent tip growth and growth oscillation in pollen tubes.To examine the signaling of protein kinases associated with LLP12-2 during germination and tube elongation, bombarded pollen was incubated in the absence or presence of okadaic acid or staurosporine. Okadaic acid is a membrane-permeable inhibitor of serine/threonine protein phosphatases types 1 and 2A,25 whereas staurosporine is a potent broad-spectrum inhibitor of serine/threonine kinases.26 The LLP12-2-overexpressing pollen did not exhibit appreciable increase in germination with the treatment of either staurosporine or okadaic acid when compared with that without treatment (Fig. 1). This implies that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2-regulated germination. Nevertheless, it is intriguing that the LLP12-2-induced inhibition of tube growth was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube elongation (Figure 2. It is consistent with the observation that a double mutation of two CDPKs severely reduces tube length but does not reduce germination.27In conclusion, we report unique desiccation-associated ABA signaling transduction through which the Rop and its target LLP12-2 are regulated during pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibits pollen germination and tube growth. An optimum level of [Ca2+]cyt influx is crucial for normal germination and tube growth. In addition, staurosporine-sensitive protein kinases also involve in the LLP12-2-induced inhibition of tube growth, but may be not involved in germination. 相似文献
Environmental and developmental signals can elicit differential activation of membrane proton (H+) fluxes as one of the primary responses of plant and fungal cells. In recent work,1 we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H+ flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H+-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H+ effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H+ fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.Key words: H+-specific vibrating probe, pH signatures, arbuscular mycorrhiza, pH signalling, Gigaspora margaritaThe 450-million-year-old symbiosis between the majority of land plants and arbuscular mycorrhizal (AM) fungi is one of the most ancient, abundant and ecologically important symbiosis on Earth.2,3The development of AM interaction starts before the physical contact between the host plant roots and the AM fungus. The hyphal growth and branching are induced by the root factors exudated by host plants, followed by the formation of appressorium leading to the hyphal penetration in the root system. These root factors seems to be specifically synthesized by host plants, since exudates from non-host plants are not able to promote neither hyphal differentiation nor appressorium formation.4,5 Most root exudates contain several host signals or better, active compounds including flavonoids6,19 and strigolactones,7,8 however many of them are not yet known.Protons (H+) may have an important role on the fungal growth and host signal perception.1 In plant and fungal cells, H+ can be pumped out through two different mechanisms: (1) the activity of the P-type plasma membrane (PM) H+-ATPase9 and (2) PM redox reactions.10 The proportional contribution from both mechanisms is not known, but in most plant cells the PM H+-ATPase seems to be the major responsible by the H+ efflux across plasma membrane. AM Fungal cells also energize their PM using P-type H+-pumps quite similar to the plant ones. Indeed, some genes codifying isoforms of P-type H+-ATPase have been isolated of AM fungi,11–13 and AM fungal ATP hydrolysis activity was shown by cytochemistry, localized mainly in the first 70 µm from the germ tube tip.14 This structural evidence correlates with data obtained by H+-specific vibrating probe (Fig. 1A and B), which indicates that the H+ efflux in Gigaspora margarita is more intense in the subapical region of the lateral hyphae1 (Fig. 1A). Furthermore, the correlation between the cytosolic pH profile previously obtained by Jolicoeur et al.,15 with the H+ efflux pattern (erythrosine-dependent), seems to clearly indicate that an active PM H+-ATPase takes place at the subapical hyphal region. Using orthovanadate, we could show that those H+ effluxes are susceptible mainly in the subapical region, but no effect in the apical was found.1 Recently, a method to use fluorescent marker expression in an AM fungus driven by arbuscular mycorrhizal promoters was published.31 It could be adjusted as an alternative to measure “in vivo” PM H+-ATPase expression in AM fungal hyphae and their responses to root factors.31Open in a separate windowFigure 1(A) H+ flux profile along growing secondary hyphae of G. margarita in the presence (open squares) or absence (closed squares) of erythrosin B and its correlation with cytosolic pH (pHc) data described by Jolicoeur et al.,15 (dotted line). Dotted area depicts the region with higher susceptibility to erythrosin B. (B) ion-selective electrode near to AM fungal hyphae. (C) Stimulation on hyphal H+ efflux after incubation with root factors or whole root system. R, roots; RE, root exudates; CO2, carbon dioxide; CWP, cell wall proteins; GR24, synthetic strigolactone. The medium pH in all treatment was monitored and remained about 5.7, including with prior CO2 incubation. Means followed by the same letter are statistically equal by Duncan''s test at p < 5%.The H+ electrochemical gradient generated by PM H+-ATPases provides not only driving force for nutrient uptake,9,16 but also can act as an intermediate in signal transduction pathways.18 The participation of these H+ pumps in cell polarity and tip growth of plant cells was recently reported,27 addressing their crucial role on apical growth.28 Naturally, in the absence of root factors the AM fungi have basal metabolic8,21–23 and respiratory activity.24 However when root signals are recognized and processed by AM fungal cells they might become activated.22 We thus searched for pH signatures that could reflect the alterations on fungal metabolism in response to external stimuli. In fact, preliminary analyses from our group demonstrate that AM fungal hyphae increase their H+ efflux in response not only to root exudates recognition, but also to other root factors (Fig. 1C). The incubation for 30 min of AM fungal hyphae with several root factors induces hyphal H+ efflux similar to the response to intact root system (5 days of incubation). The major increases were found with 1% CO2 (750%) followed by root cell wall proteins (221%), root exudates (130%) and synthetic strigolactone (5%) (Fig. 1C). Those stimulations could define the transition from the state without root signals to the presymbiotic developmental stage (Fig. 1C). In the case of CO2, the incorporation of additional carbon could represent a new source of energy, since CO2 dark fixation takes place in Glomus intraradices germ tubes.22,25Interestingly, after the treatment with synthetic strigolactone (10−5 M GR24), no significant stimulation was found compared to the remaining factors (Fig. 1C). It opens the question if the real effect of strigolactone is restrict to hyphal branching and does not intervene in very fast response pathways. Likewise, strigolactones need additional time to exhibit an effect, as recently discussed by Steinkellner et al.,26 However, at the moment, no comprehensive electrophysiological analyses are presently available separating the effects of strigolactone and some flavonoids in AM fungal hyphae.The next target of our work is the study of ionic responses of single germ tubes or primary hyphae to root factors (Fig. 2). As reported by Ramos et al.,1 we have been observing that the pattern of ion fluxes at the apical zone of primary hyphae is differentiated from secondary or lateral hyphae. In the primary, two interesting responses were detected in the absence of root factors: (1) a “dormant Ca2+ flux” and (2) Cl− or anion fluxes at the same direction of H+ ions, suggesting a possible presence of H+/Cl− symporters at the apex, similarly to what occurs in root hairs (Fig. 2).30 In the presence of root factors such as root exudates the stimulated influxes of Cl− (anion), H+, Na+ and effluxes of K+ and Ca2+ are activated. It can explain why the AM fungi hyphal tips are depolarized20,29 during the period without root signals—“asymbiosis”—as long as K+ efflux and H+ influx occur simultaneously. Indeed, H+ as well as Ca2+ ions may act as second messengers, where extra and intracellular transient pH changes are preconditions for a number of processes, including gravity responses and possibly in plant-microbe interactions.17,30Open in a separate windowFigure 2Ion dynamics in the apex of primary hyphae of arbuscular mycorrhizal fungi. It represents the Stage 1 described in Ramos et al.1 After treatment with root factors, an activation of Ca2+ efflux is observed at the hyphal apex.Clearly, further data on the mechanism of action of signaling molecules such as strigolactones over the signal transduction and ion dynamics in AM fungi will be very important to improve our understanding of the molecular bases of the mycorrhization process. Future studies are necessary in order to provide basic knowledge of the ion signaling mechanisms and their role on the response of very important molecules playing at the early events of AM symbiosis. 相似文献
The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4+ T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3, and E4 regions do not induce appreciable expansion of vector-specific CD4+ T cells.Replication-defective adenoviruses (rAd) have been engineered to provide high levels of expression of foreign inserts with minimum expression of adenovirus proteins, making them excellent candidates for vaccine and gene therapy applications (3, 16). Despite promising immunogenicity, a prophylactic vaccine trial of a serotype 5 rAd (rAd5) vector expressing human immunodeficiency virus (HIV) Gag, Pol, and Nef genes (Step trial) was recently halted due to an increase in HIV infections among volunteers who had preexisting neutralizing antibodies (NAs) to Ad5 (7). This finding raises the possibility that the presence of Ad5-specific T-cell responses (specifically CD4+ T-cell responses) in subjects with preexisting Ad5 NAs could be boosted by rAd5 vaccines, thereby providing an expanded susceptible target cell population that could be more easily infected by HIV. If this mechanism were operative, it would have broad implications for the future use of rAd viruses, and indeed other virus vectors, as vaccines or therapeutic agents within HIV-susceptible populations (2, 12, 15). We therefore measured the frequency, magnitude, and activation status of rAd5-specific T cells in HIV-uninfected volunteers who had received rAd5-based HIV vaccines in the presence or absence of preexisting NAs to Ad5.We studied 31 volunteers enrolled in two NIAID Institutional Review Board-approved phase I clinical trials of rAd5-based HIV vaccines. VRC 006 was a dose escalation study evaluating a single inoculation of a rAd5 mixture expressing EnvA, EnvB, EnvC, and fusion protein Gag/PolB at 109, 1010, and 1011 total particle units (10). VRC 008 evaluated DNA priming by needle and syringe or Biojector, followed by rAd5 boosting. Both studies enrolled healthy, HIV-uninfected adults; used the same rAd5 products; and evaluated immunogenicity on the day of and 4 weeks after rAd5 immunization. Both of these trials involved rAd5 products that contained deletions in the E1, E3, and E4 regions (8, 10).NAs to Ad5 were determined for all volunteers as previously described (19). A 90% NA titer of 12 or more was considered positive and taken as evidence of preexisting humoral immunity to Ad5. Volunteers were chosen for assessment of Ad5-specific T-cell responses based upon the availability of peripheral blood mononuclear cell samples at key time points and the presence or absence of preexisting NAs to Ad5. Only volunteers who received the vaccine (not the placebo) were included. Table Table11 lists the volunteers who were tested for Ad5-specific T-cell responses and their NA titers to Ad5 before and after rAd5 vaccination. All volunteers, except for one (volunteer 12) who had a less-than-maximum NA titer to Ad5 before vaccination, had an increase in titer by 4 weeks after vaccination, indicating the successful “take” of the rAd5-based vaccine. There was no correlation between rAd5 dose and increase in Ad5 NA titer.
TABLE 1.
Ad5 serostatus before and after vaccination
Volunteer
Prior DNA immunization
rAd5 dose (PUa)
Ad5 NA titer
Prevaccine
Postvaccine
1
No
1011
<12
739
2
No
1011
<12
834
3
No
1011
<12
4,787
4
No
1011
<12
806
5
No
1011
<12
1,033
6
No
1010
<12
130
7
No
1010
<12
1,354
8
Yes
1010
<12
1,387
9
Yes
1010
<12
575
10
Yes
1010
<12
170
11
Yes
1010
<12
>8,748
12
Yes
1010
<12
<12
13
No
1011
30
>8,748
14
No
109
46
>8,748
15
No
109
70
328
16
No
1010
176
>8,748
17
No
1010
478
6,198
18
No
109
2,472
>8,748
19
No
109
3,502
>8,748
20
No
1010
4,820
>8,748
21
No
109
5,078
>8,748
22
No
1011
6,162
>8,748
23
No
109
>8,748
>8,748
24
No
1011
>8,748
>8,748
25
Yes
1010
643
>8,748
26
Yes
1010
942
>8,748
27
Yes
1010
1,510
>8,748
28
Yes
1010
1,611
>8,748
29
Yes
1010
2,934
>8,748
30
Yes
1010
>8,748
>8,748
31
Yes
1010
>8,748
>8,748
Open in a separate windowaPU, particle units.HIV-specific T-cell responses were measured by multiparameter flow cytometry after 6 h of stimulation with peptides (15-mers overlapping by 11) corresponding to the HIV EnvA protein (one of the vaccine inserts expressed in the Ad5 vectors), as previously described (13). Overlapping peptides corresponding to the major Ad5 surface protein (hexon), the Ad5 early regulatory protein (E2A), and Ad5 ORF1, -2, and -3 proteins were used to assess Ad5-specific T-cell responses, and additional markers of cell viability (ViViD), T-cell memory (CD45RO and CD27), and activation/division (CCR5, CD38, HLA-DR, and Ki67) were added to the panel for these assessments. Antibodies and fluorochromes used in this panel were CCR5-Cy7-phycoerythrin (PE), CD38-allophycocyanin, Ki67-fluorescein isothiocyanate, and CD3-Cy7-allophycocyanin, all from BD PharMingen; CD8-Cy55-PE from BD Biosciences; CD27-Cy5-PE and CD45RO-Texas Red-PE, both from Beckman Coulter; CD4-Cy5.5-PE from Caltag; CD14- and CD19-PacificBlue, CD57-QDot545, and HLA-DR-Alexa680, conjugated according to standard protocols [http://drmr.com/abcon/index.html]); gamma interferon-PE and interleukin-2-PE from BD Biosciences; and a violet amine dye from Invitrogen. Cells were analyzed on an LSRII instrument (Becton Dickinson), and data analysis was performed using FlowJo, version 8.1.1 (TreeStar). The gating strategy is shown in Fig. Fig.1A1A.Open in a separate windowFIG. 1.CD4+ and CD8+ T-cell responses to Ad5. (A) Gating tree used to determine antigen-specific T-cell frequencies. Single CD3+ ViViD− CD14− CD19− cells were gated on CD4 or CD8 cells. Naïve CD27+ CD45RO− cells were gated out, and the frequency of cells expressing gamma interferon (IFNg) and/or interleukin-2 (IL2) was determined. FSC-A, forward scatter area; FSC-H, forward scatter height; SSC-A, side scatter area. (B) Frequencies of CD4+ and CD8+ T-cell responses after stimulation with Ad5 hexon or E2A peptides were plotted against the prevaccination Ad5 NA titer. The prevaccine T-cell response was used. (C) Frequencies of CD4+ and CD8+ T-cell responses to Ad5 hexon, E2A, and HIV EnvA before and 4 weeks after rAd5 vaccination are shown for subjects with (Ad5 NA titer of >12) and without (Ad5 NA titer of <12) preexisting NAs to Ad5. Boxed areas represent interquartile ranges, and horizontal lines represent medians.Previously, we had found no T-cell responses to Ad5 ORF1, -2, or -3, so data from these antigen stimulations are not shown. As shown in Fig. Fig.1B,1B, T-cell responses to Ad5 hexon and E2A were detected, but there was no association between the NA response to Ad5 and the T-cell responses to these Ad5 proteins. Volunteers with an absence of NAs to Ad5 often had very strong CD4+ and CD8+ T-cell responses to Ad5 proteins. This probably reflects the degree of protein sequence homology between different adenovirus serotypes (11) and suggests that T-cell responses to adenoviruses may be significantly cross-reactive, while NAs are serotype specific. It also indicates that the NA response to Ad5 cannot be used as a surrogate for either a CD4+ or a CD8+ T-cell response to that adenovirus serotype.We next asked whether Ad5-specific T-cell responses were boosted by a single rAd5 vaccination in subjects with or without preexisting NAs to Ad5. At the time point 4 weeks after vaccination, there was clear evidence of boosting of the insert-specific (EnvA) CD4+ and CD8+ T-cell responses in volunteers with and without preexisting NAs to Ad5 (Fig. (Fig.1C).1C). The results of the Ad5-specific responses were consistent across volunteers who had received prior DNA immunization (VRC 008) and those who had not (VRC 006), so the results are combined in Fig. Fig.1C1C and show no increase in Ad5 hexon- or E2A-specific CD4+ T-cell responses after rAd5 immunization irrespective of Ad5 NA status. There was evidence of an increase in the CD8+ T-cell response to Ad5 hexon (P = 0.004 by paired t test), but not that to E2A, after rAd5 vaccination. These results, while showing evidence of adenovirus-specific CD8+ T-cell boosting by rAd5 vaccination, do not indicate an expansion of Ad5-specific CD4+ T cells that could serve as a substrate for HIV infection in subjects with or without NAs to Ad5.Having failed to demonstrate an expansion of Ad5-specific CD4+ T cells after vaccination, we assessed whether the activation profile of the unexpanded Ad5-specific CD4+ T cells was changed by vaccination. The gating tree is shown in Fig. Fig.2A.2A. Ad5 hexon- and E2A-specific CD4+ T cells expressed activation markers CCR5, CD38, and HLA-DR and a marker of recent cell division, Ki67, more frequently than did total memory CD4+ T cells (Fig. (Fig.2B).2B). However, none of these markers were significantly increased on total or Ad5-specific CD4+ T cells after vaccination in volunteers with or without preexisting NAs to Ad5.Open in a separate windowFIG. 2.Vaccine-induced activation of Ad5-specific CD4+ T cells. (A) Total CD4+ memory cells or Ad5-specific CD4+ memory cells (as gated in Fig. Fig.1A)1A) were further defined by expression of Ki67, CD38, CCR5, and HLA-DR. (B) Percentages of Ad5 hexon-specific cells, E2A-specific cells, or total memory CD4+ T cells that express CCR5, CD38, HLA-DR, or Ki67 before and 4 weeks after rAd5 vaccination are shown for subjects with (Ad5 NA titer of >12) (left) and without (Ad5 NA titer of >12) (right) preexisting NAs to Ad5. The phenotype was assessed only for those responders for whom at least 10 cytokine-positive events were counted. None of the comparisons of pre- and postvaccination marker expression were significant at a P value of 0.02 by paired t test. Boxed areas represent interquartile ranges, and horizontal lines represent medians.Expansion of Ad5-specific T cells after rAd5-based vaccination or gene therapy has been reported by others (14, 20, 21). Those studies evaluated Ad5-specific responses to rAd5 vectors with only the adenovirus E1 gene deleted (as used in the Step trial vaccines). The vectors used here contained deletions of the adenovirus E1, E3, and E4 genes (8, 10). While adenovirus gene deletions can render the vectors replication defective (6, 9), they do not necessarily completely shut off all adenovirus protein expression (20, 21). To demonstrate the importance of E4 deletions in limiting expression of adenovirus gene products, we measured the level of adenovirus protein synthesis in infected A549 cells as previously described (1, 4, 5). Cells were infected with adenovirus vectors with E1 and E3 deletions or with E1, E3, and E4 deletions at the same multiplicity of infection (10 focus-forming units per cell). At 24 h postinfection, [35S]methionine was added for 1 h. Levels of total and adenovirus protein synthesis in the infected and mock-infected cells were compared (Fig. (Fig.3).3). Adenovirus early protein single-stranded DNA binding protein, as well as late gene products hexon, penton, and fiber, was immunoprecipitated, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and resolved by autoradiography. The results show that the amount of newly synthesized adenovirus proteins in cells infected with adenovirus with E1, E3, and E4 deletions is significantly lower than that for an adenovirus vector with E1 and E3 deletions. Therefore, our inability to detect a vaccine-induced increase in the frequency and character of the Ad5-specific T-cell response could relate to the very low levels of adenovirus proteins that were probably expressed in vivo by the rAd5 vectors with multiple deletions.Open in a separate windowFIG. 3.Ad5 protein expression in vitro after infection with different Ad5 vectors. A549 cells were infected with adenovirus vectors with E1 and E3 deletions or with E1, E3, and E4 deletions and [35S]methionine labeled, and levels of total and adenovirus protein synthesis in the infected and mock-infected cells were compared after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Markers for the adenovirus early protein single-stranded DNA binding protein (DBP) and capsid proteins hexon, penton base, and fiber are shown.We were therefore unable to demonstrate (i) that Ad5-specific CD4+ T cells were restricted to subjects with preexisting Ad5 NAs, (ii) that rAd5 vaccination expanded or increased the activation of Ad5-specific CD4+ T cells, or (iii) that there was a substantial effect on the magnitude or character of the Ad5-specific CD4+ T-cell response to vaccination based upon preexisting NAs to Ad5. While the kinetics of Ad5-specific T-cell responses after rAd5-based vaccination are not known, it is clear that insert-specific responses are increased at 4 weeks after vaccination and subsequently contract (10). It is therefore reasonable to assume that if Ad5-specific responses were similarly affected, they would be detected at the 4-week-postvaccination time point.It is possible that rAd5 vaccines expand a preexisting mucosal T-cell response to Ad5 that is not reflected within the blood. While we do not have mucosal samples from our vaccine volunteers to directly address this possibility, it is likely that expansion of a mucosal response would be reflected to some degree within the blood.The mechanism underlying the increase in HIV infections in vaccinees with NAs to Ad5 in the Step trial is yet to be determined (2, 7, 12, 15, 17). Confounding factors and alternative hypotheses have recently been proposed to account for the increased acquisition (7, 12, 15, 18). Until there is a better understanding of the processes involved, future studies of rAd5-based products should proceed with appropriate safety considerations and monitoring of adenovirus-specific responses. In addition, the use of vaccine regimens involving single injections of vectors with multiple deletions may help mitigate risk. 相似文献
The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.Key words: cell wall invertase, apoplastic invertase, alkaline invertase, neutral invertase, sucrose synthase, plant defense, Nicotiana tabacum, Phytophthora nicotianaePlant defense against pathogens is costly in terms of energy and carbohydrates.1,2 Sucrose (Suc) and its cleavage products glucose and fructose are central molecules for metabolism and sensing in higher plants (reviewed in refs. 3 and 4). Rapid mobilization of these carbohydrates seems to be an important factor determining the outcome of plant-pathogen interactions. In particular in source cells reprogramming of the carbon flow from Suc to hexoses may be a crucial process during defense.1,2There are two alternative routes of sucrolytic carbohydrate mobilization. One route is reversible and involves an uridine 5′-diphosphate (UDP)-dependent cleavage catalyzed by sucrose synthase (SUSY). Its activity is limited by the concentrations of Suc and UDP in the cytosol, as the affinity of the enzyme to its substrate is relatively low (Km for Suc 40–200 mM). The other route is the irreversible, hydrolytic cleavage by invertases (INVs), which exhibit high affinity to Suc (Km 7–15 mM).5Plants possess three different types of INV isoenzymes, which can be distinguished by their solubility, subcellular localization, pH-optima and isoelectric point. Usually, they are subdivided into cell wall (cwINV), vacuolar (vacINV), and alkaline/neutral (a/nINVs) INVs.cwINV, also referred to as extracellular or apoplastic INV, is characterized by a low pH-optimum (pH 3.5–5.0) and usually ionically bound to the cell wall. It is the key enzyme of the apoplastic phloem unloading pathway and plays a crucial role in the regulation of source/sink relations (reviewed in refs. 3, 6–8). A specific role during plant defense has been suggested, based on observations that cwINV is often induced during various plant-pathogen interactions, and the finding that overexpression of a yeast INV in the apoplast increases plant resistance.6,8–10 It was shown, that a rapid induction of cwINV is, indeed, one of the early defense-related reactions in resistant tobacco source leaves after infection with Phytophthora nicotianae (P. nicotianae).11 Finally, the whole infection area in wt leaves was covered with hypersensitive lesions, indicating that all cells had undergone hypersensitive cell death (Fig. 1A).1,11 When the activity of cwINV was repressed by an RNAi construct, defense-related processes were impaired, and the infection site exhibited only small spots of hypersensitive lesions. Finally, the pathogen was able to sporulate, indicating a reduced resistance of these transgenic plants (Fig. 1A).1Open in a separate windowFigure 1Defense-induced changes in the activity of intracellular sucrose-cleaving enzymes and their contribution to defense. (A) The repression of cwINV in source leaves of tobacco leads to impaired pathogen resistance and can not be compensated by other sucrose-cleaving enzymes. The intensity of defense reactions is amongst others indicated by the extent of hypersensitive lesions. (B and C) Absolute activity of vacuolar (B) and alkaline/neutral (C) INVs at the infection site (white symbols, control; black symbols, infection site). (D) Increase in SUSY activity at the infection site. All data points taken from noninfected control parts of the plants in each individual experiment and each point along the time scale of an experiment are set as 0%. At least three independent infections are averaged and their means are presented as percentage changes ± SE (circles, SNN; triangles, SNN::cwINV). Insets show the means of the absolute amount of activities (white symbols, control; black symbols, infection site). Material and methods according to Essmann, et al.1vacINV, also labeled as soluble acidic INV, is characterized by a pH optimum between pH 5.0–5.5. Among others it determines the level of Suc stored in the vacuole and generates hexose-based sugar signals (reviewed in refs. 3 and 12). Yet, no specific role of vacINV during pathogen response has been reported. Although vacINV and cwINV are glycoproteins with similar enzymatic and biochemical properties and share a high degree of overall sequence homology and two conserved amino acid motifs,4 the activity of vacINV in tobacco source leaves was not changed due to the repression of the cwINV (Fig. 1B).1 After infection with P. nicotianae the activity of vacINV in wt SNN did not respond under conditions where cwINV was stimulated.1 There was also no significant change in the transgenic SNN::cwINV (Fig. 1B). This suggests that during biotic stress, there is no crosstalk between the regulation of cwINV and vacINV.a/nINVs exhibit activity maxima between pH 6.5 and 8.0, are not glycosylated and thought to be exclusively localized in the cytosol. But recent reports also point to a subcellular location in mitochondria and chloroplasts.13,14 Only a few a/nINVs have been cloned and characterized, and not much is known about their physiological functions (reviewed in refs. 4, 14 and 15). Among other things they seem to be involved in osmotic or low-temperature stress response.14,15 During the interaction between tobacco and P. nicotianae the activity of a/nINVs rose on average 17% in the resistant wt SNN between 1 to 9 hours post infection (Fig. 1C). By contrast, in SNN::cwINV the a/nINVs activities remained unchanged in control leaves and even after infection (Fig. 1C). This suggests that the defense related stimulation in a/nINVs activities is rather a secondary phenomenon, possibly in response to the enhanced cwINV activity and the related carbohydrate availability in the cytosol.SUSY can be found as a soluble enzyme in the cytosol, bound to the inner side of the plasma membrane or the outer membrane of mitochondria, depending on the phosphorylation status. It channels hexoses into polysaccharide biosynthesis (i.e., starch, cellulose and callose) and respiration.12,16 There is also evidence that SUSY improves the metabolic performance at low internal oxygen levels17 but little is known about its role during plant defense. Callose formation is presumably one of the strongest sink reactions in plant cells.1,18 Defense-related SUSY activity may serve to allocate Suc into callose deposition and other carbohydrate-consuming defense reactions. In fact, in the resistant wt the activity of SUSY increased upon interaction with P. nicotianae in a biphasic manner (Fig. 1D). The time course is comparable to that of cwINV activity and correlates with callose deposition and enhanced respiration.1,11 However, repression of cwINV leads in general to a reduction of SUSY activity in source leaves of tobacco.1 After infection the activation of SUSY was also significantly impaired (Fig. 1D). At the same time, the early defense-related callose deposition in infected mesophyll cells of SNN::cwINV plants is substantially delayed.1 It is known that expression of SUSY isoforms is differentially controlled by sugars,12 and there is evidence that hexoses generated by the defense-induced cwINV activity deliver sugar signals to the infected cells.1 In this sense, the reduction of defense-related, cwINV-generated sugar signals could be responsible for the repression of SUSY activity in SNN::cwINV plants after infection with P. nicotianae.Only limited hexoses or hexose-based sugar signals could be generated by cytoplasmic Suc cleavage.12 The reduction of soluble carbohydrates for sugar signaling and also as fuel for metabolic pathways that support defense reactions could be responsible for the impaired resistance in SNN::cwINV plants (Fig. 1A).Obviously, neither intracellular INV isoforms, nor SUSY can compensate for the reduced carbohydrate availability due to cwINV repression during plant defense. The data also suggest that the activity of SUSY is affected by cwINV and related reflux of carbohydrates. It is known that SUSY activity can be controlled, e.g., by sugar-mediated phosphorylation12 and one may speculate that posttranslational modulation of the protein is affected by the defense-related carbohydrate status of the cell. 相似文献