The mcyB, aerB, and apnC genes occurred in

99%, 99%, and 97% of the samples, respectively, and on average comprised 60 ± 3%, 22 ± 2%, and 54 ± 4% of the total population, respectively. Although the populations differed widely in abundance (10−3–103 mm3 L−1) no dependence of the proportion of the mcyB, aerB, and apnC genes on the density of the total population was found. In contrast populations differed significantly in their average mcyB, aerB, and apnC gene proportions, with no change between prebloom and bloom conditions. These results emphasize stable population-specific differences in mcyB, aerB, and apnC proportions that are independent from seasonal influences. “
“Antimicrobial peptides (AMPs) are present in virtually all organisms selleckchem and are an ancient and critical component of innate immunity. In mammals, AMPs are present in phagocytic cells, on body surfaces such as skin and mucosa, and in secretions and selleck screening library body fluids such as sweat, saliva, urine,

and breast milk, consistent with their role as part of the first line of defense against a wide range of pathogenic microorganisms including bacteria, viruses, and fungi. AMPs are microbicidal and have also been shown to act as immunomodulators with chemoattractant and signaling activities. During the co-evolution of hosts and bacterial pathogens, bacteria have developed the ability to sense and initiate an adaptive response to AMPs to resist their bactericidal activity. Here, we review the various mechanisms used by Gram-negative bacteria to sense and resist AMP-mediated killing. These mechanisms play an important role in bacterial resistance to host-derived AMPs that are encountered during the course of infection. Bacterial resistance to AMPs should also be taken into consideration in the

development and use of AMPs as anti-infective agents, for which there is currently a great deal of academic and commercial interest. Mammalian antimicrobial peptides (AMPs) are diverse Terminal deoxynucleotidyl transferase in sequence and are classified into families on the basis of their structures and functions (Hancock & Sahl, 2006). Two major families of AMPs in mammals are the defensins and the cathelicidins (Table 1). Defensins are cysteine-rich cationic peptides that form β-sheet structures and contain disulfide bonds. The position of the disulfide bonds is used to further classify defensins into subfamilies (α- and β-defensins in mice and humans). Of note, murine α-defensins are often designated as cryptdins (Eisenhauer et al., 1992). Cathelicidins are also positively charged, but do not have disulfide bonds. Rather, they form amphipathic α-helices with a positively charged face. There is only one cathelicidin member present in humans and mice, named LL-37 and murine cathelicidin-related antimicrobial peptide (mCRAMP), respectively.

, 2009). The analysis of RepB from pPRH revealed one conserved domain homologous to region 4 of sigma-70-like sigma factors, which is involved TSA HDAC in binding of the −35 promoter element (Campbell et al., 2002). The RepB protein of pAL5000 was shown to bind to DNA near the ori site (Stolt & Stoker, 1996b). It could be proposed that the RepB encoded by pPRH has

the same function. According to the sequence analysis, ORF6 belongs to serine recombinase family, which includes resolvases, invertases, integrases and transposases (Smith & Thorpe, 2002), and might contribute to plasmid maintenance (Nordstrom & Austin, 1989). A putative resolvase of plasmid pPRH is phylogenetically most related to the enzyme from A. arilaitensis sharing the distinct branch (Fig. 2c). This demonstrates that, in contrast to both Rep proteins, the resolvase displays the independent patterns of evolution. Escherichia coli–Arthrobacter–Rhodococcus shuttle vectors were built using the bottom-up approach, starting with the minimal requirement for the arthrobacterial replicon taken from the cryptic plasmid pPRH. The multiple cloning site of the lacZ′ cassette

(Fig. 3) allowed using a common beta-galactosidase-based screening strategy in E. coli. The developed shuttle vectors were compatible with the pART vectors (Sandu et al., 2005). Hence, these plasmids might be used as original tools in genetic complementation studies as well as for a functional complementation-based screening in both Arthrobacter and Rhodococcus species. The successful cloning of the genes encoding the initial steps of 2-hydroxypyridine biodegradation in Arthrobacter sp. PY22 Ponatinib nmr showed a potential of the developed vectors for functional screening in the nonconventional host. The cloned genes or encoded proteins were inactive in E. coli cells; hence, screening based on enzyme activities was impossible in this host. However, the pHYP1 plasmid containing genes encoding 2-hydroxypyridine catabolism could be selected using Rhodococcus or Arthrobacter as a host. It is supposed that 2-hydroxypyridine biodegradation in Arthrobacter sp. PY22 bacteria proceeds

via classical pathway by formation of 2,5-dihydroxypyridine and 2,3,6-trihydroxypyridine as intermediates (Semėnaitė et al., 2003). Implying that, the appropriate hydroxylases are expected. A sequence analysis of the cloned DNA fragment Temsirolimus chemical structure showed that hpyB gene encodes a putative flavin monooxygenase belonging to the family of flavin mononucleotide (FMN)-dependent bacterial luciferases and alkanesulphonate monooxygenases, enzymes that employ reduced flavin and usually act as two-component monooxygenases in concert with NAD(P)H-dependent FMN reductases (Ellis, 2010). The hpyD gene encoding a putative NAD(P)H-dependent FMN reductase is located in close proximity to the hpyB gene. Hence, a two-component flavin monooxygenase involved in the hydroxylation of 2-hydroxypyridine ring might be expected.

Data suggest that ART can be delayed until the first 2 months of TB therapy has been completed but at CD4 cell counts <50 cells/μL the short-term risk of developing further AIDS-defining events this website and death is high, and ART should be started as soon as practicable and within 2 weeks of initiation of TB therapy [2-5]. Starting ART early in severely immunosuppressed HIV-positive patients presenting with TB is associated with decreased

mortality and a lowering of the rates of disease progression but rates of IRD are high. Patients with HIV and a CD4 cell count >350 cells/μL have a low risk of HIV disease progression or death during the subsequent 6 months of TB treatment, depending on age and VL [6]. They should have their CD4 cell count monitored regularly and ART can be withheld during the short-course of TB treatment. One study performed in HIV-associated selleck products TB meningitis in the developing world, where 90% of the patients were male, the majority drug users, many with advanced disease and the diagnosis being made clinically, showed no difference in mortality starting ART early or

late [7]. We recommend EFV in combination with TDF and FTC as first-line ART in TB/HIV coinfection 1B We recommend that when rifampicin is used with EFV in patients over 60 kg, the EFV dose is increased to 800 mg daily. Standard doses of EFV are recommended if the patient weighs <60 kg 1C We recommend that rifampicin is not used with either NVP or PI/r 1C We recommend that where effective ART necessitates the use of PI/r, that rifabutin is used instead of rifampicin 1C Proportion of patients with active TB on anti-TB therapy why started on ART containing EFV, TDF and FTC. HIV-related TB should be treated with a regimen, including rifamycin for the full course of TB treatment, unless there is rifamycin resistance or intolerance. Rifamycins frequently interact with ARV medications and can lead to similar toxicities, notably rash and hepatitis. We recommend EFV as the preferred therapy for ART because of its confirmed potency when used in TB/HIV

coinfection [8-10], and its efficacy in RCT. We recommend that EFV be given with TDF and FTC due to the availability of a once-daily co-formulation, a reduced risk of rash compared with NVP and improved efficacy at higher HIV VLs (commonly occurring in this setting). ABC-3TC is an alternative acceptable NRTI backbone in patients with lower HIV VLs and that are HLA-B*57:01 negative (see Section 5.3 Which NRTI backbone). There is significant variability in the effect that rifampicin has on EFV concentrations because of liver enzyme induction, especially of CYP450 3A4 [8,11–13]. Subtherapeutic EFV concentrations may occur among patients who weigh more than 60 kg who are taking standard dose EFV together with rifampicin, and increasing the dose of EFV from 600 mg daily to 800 mg daily may be necessary; however, there is a risk of increasing adverse effects.

Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the

view that high temperature negatively affects Target Selective Inhibitor Library ic50 aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster. Aspergillus flavus produces aflatoxins B1 and B2 and causes aflatoxin contamination of preharvest crops such as corn, cotton, peanuts and tree nuts, and postharvest grains during storage (Bhatnagar et al., 1987; Bennett & Klich, 2003). The discovery of the first stable aflatoxin precursor, norsolorinic acid (Bennett, 1981), paved the way

for the elucidation of the aflatoxin biosynthetic pathway, including its intermediates and biosynthetic gene clusters in A. flavus, Aspergillus parasiticus, Aspergillus nidulans (sterigmatocystin as end product), Aspergillus sojae and Aspergillus oryzae (nonfunctional gene cluster) (Brown et al., 1996; Yu et al., 2004a, b). Aflatoxin biosynthesis is affected by many biotic and abiotic factors (Payne & Brown, 1998; Yu et al., 2010). The influence of temperature eltoprazine on aflatoxin formation has been reported previously (Schroeder & Hein, 1968; Ogundero, 1987). The optimum MK-2206 in vivo temperature for biosynthesis of aflatoxin and other secondary metabolites is at 30 °C; while the optimum temperature for fungal growth is at about 37 °C but it is less optimal for mycotoxin production. Sequencing of the A. flavus genome facilitated the construction of microarrays, which have been used to study transcriptional

regulation of aflatoxin biosynthesis at different temperatures (OBrian et al., 2007; Georgianna et al., 2008, 2010; Payne et al., 2008; Schmidt-Heydt et al., 2009). These studies identified a large number of genes expressed at high level under low temperature. The effect of temperature on natural antisense transcript expression was also reported (Smith et al., 2008). While microarrays are a robust tool for genome-wide gene expression analysis, they have been plagued by high background and low sensitivity problems. For regulatory genes with low level of expression, microarrays often fail to provide meaningful information about their expression levels. Thus, no published microarray experiments have provided an accurate estimate of the aflR and aflS expression levels. RNA-Seq technology has been successful for transcriptome profiling in a closely related species, A. oryzae (Wang et al., 2010).

6xHIS and Δcox15 with ScCOX15.6xHIS, as positive controls. Using two different expression vectors (see Materials and methods), the same phenotype suppression was observed, demonstrating that T. cruzi sequences are able to complement yeast respiratory deficiencies. To confirm these results, the oxygen consumption of WT, Δcox10, Δcox15 yeast strains and their corresponding transformants was measured (Fig. 2b). As expected, the knockout cells were impaired in O2 consumption due to their inability to produce heme A and consequently fully active CcO. The respiratory function was restored selleck products with the expression of the corresponding T. cruzi COX10 and COX15

genes, as well as with the S. cerevisiae COX10 and COX15 genes. Taken together, these results demonstrate that TcCOX10 and TcCOX15 encode HOS and HAS enzymes that are functional in the yeast model. In order to verify the function of these proteins in heme A biosynthesis, the mitochondrial heme level was evaluated by differential absorption spectroscopy as described previously (Tzagoloff et al., 1975). The reduced minus oxidized spectra of mitochondrial cytochromes were recorded and are presented in Fig. 3a. The spectra of the knockout

cells only exhibited signals corresponding to heme b and heme c, and the heme a signal was absent, confirming the deficiency of its biosynthesis (Nobrega et al., 1990; Glerum et al., 1997). The spectrum recorded from the mitochondria of WT cells displayed bands corresponding to heme a, heme b

and heme c. The expression of TcCOX10 in Δcox10 and TcCOX15 in Δcox15 allowed the recovery of the heme a signal, reflecting the role in heme A synthesis of the TcCox10 and TcCox15 proteins selleck screening library as HOS and HAS enzymes, respectively. The protein levels of Cox10 and Cox15 were evaluated using Western blot analysis of yeast mitochondria. All these proteins (from S. cerevisiae and T. cruzi) were expressed as C-terminal his-tag fusion proteins (Fig. 3b). As expected, the proteins were detectable in the cells transformed with the plasmids expressing TcCOX10.6xHIS, Rutecarpine ScCOX10.6xHIS, TcCOX15.6xHIS and/or ScCOX15.6xHIS, and they were not detectable in the WT, Δcox10 or Δcox15 cells transformed with control vectors. The signals detected at around 38–45 kDa were consistent with the apparent molecular weight expected for TcCox10 and TcCox15 proteins based on their primary sequences (for TcCox10 388 aa, 42 kDa and for TcCox15 396 aa, 44 kDa, both molecular weights were estimated for the preprotein without the C-terminal tag, TriTrypDB, http://tritrypdb.org/tritrypdb/). In both cases, the band intensity of the T. cruzi proteins was always lower compared with the S. cerevisiae ones. Several factors could be involved in this observation: (1) the different mitochondrial targeting sequence [shorter in trypanosomatids (Hausler et al., 1997)] resulted in less efficient mitochondrial importation; (2) the lower stability of the T. cruzi proteins compared with the S.

More tourists presented for diarrhea than residents: 1,397 Gefitinib supplier (33%) versus 766 (16%) (relative risk 1.99; 95% CI 1.85–2.16, p < 0.001). In total, 390 cases and 185 controls

were enrolled with 381 cases and 176 controls eligible for analysis. Eighteen persons were excluded from the analysis due to incomplete information, wrong nationality, or inability to submit a stool sample. The mean age for cases was significantly younger than that for controls: 33.4 years (SD = 11.4, median = 30) versus 40.4 years (SD = 12.0, median = 39) (p < 0.001). There was no difference in gender with 203 (53%) female cases versus 101 (57%) female controls (p = 0.36). Most enrollees were from Europe (64% of cases vs 57% of controls), with the remainder from North America (25 and 32%, respectively), Australia/New Zealand (6 and 10%), and Japan (5 and 1%) (p = 0.02). More cases (56%) than controls (37%)

were tourists (p < 0.001). More cases had been in Nepal for <30 days than controls (p = 0.001). A significant portion of cases, n = 53 (14%), and just three controls click here had taken an FQ prior to presentation. The likelihood of identifying a bacterial pathogen was less if the patient reported taking an FQ [odds ratio (OR) = 0.38, p = 0.003], whereas no significant association was observed if the patient reported taking an antimotility drug or other medication. The likelihood of identifying a bacterial pathogen was greater if the patient reported watery diarrhea (OR = 2.04, p = 0.022), fever (OR = 1.84, p = 0.004), and microscopic white blood cells (WBCs) and red blood cells (RBCs) when found in stool (OR = 3.35 and 4.24, respectively, p < 0.001). Seasonality did not affect finding of bacterial pathogens (Table 1). Reported use of an Epothilone B (EPO906, Patupilone) antimotility drug was significantly associated for finding a viral pathogen (OR = 4.24, p = 0.001) and if the patient reported vomiting (OR = 2.99, p = 0.004). Viral pathogens were less likely to be found in the months of April to June (OR = 0.26, p = 0.037).

Cases in whom a protozoan pathogen was found were less likely to report sudden onset of diarrhea (OR = 0.27, p < 0.001) or abdominal pain (OR = 0.37, p = 0.001) and less likely that microscopic WBCs and RBCs were found in stool (OR = 0.42, p = 0.001 and OR = 0.31, p = 0.008, respectively). Interestingly, all pathogens, particularly protozoa, were more likely to be found in April to September, and Japanese were more likely than any other nationality to have protozoan pathogens (p = 0.009). At least one enteric pathogen was identified in 263 of 381 (69%) cases and 47 of 176 (27%) controls (p≤ 0.001; Table 2). Cases were 12 times more likely to have multiple pathogens detected than controls (p < 0.001). Among cases, multiple pathogens were more common among tourists (32%) than residents (18%) (p = 0.002). Campylobacter was the most prevalent pathogen isolated in cases (17%) and the second most common among controls (5%; p = 0.002).

The patient was, therefore, admitted to our hospital for treatment, given intravenous infusions and observed for dengue warning signs. The patient’s platelet count was at its lowest on day 7 after onset

of disease (48 × 109/L) and her fever subsided on day 8 after onset. She was discharged after hospitalization for a total of 7 days. DENV-3 genome was detected by real-time polymerase chain reaction (RT-PCR, Applied Biosystems, USA) and virus isolated using the Aedes albopictus mosquito cell line C6/36.[3] Although tests for anti-dengue IgM (Focus Diagnostics, USA), and IgG (Panbio, Australia) antibodies were negative on day 2 after onset of disease, tests using serum sample from day 8 after AZD2281 onset of disease was positive. Both day 2 and day 8 serum samples were positive for dengue NS1 antigen (Platelia, Selleck Panobinostat Bio-Rad, France). Serum samples were de-identified prior to being used in the experiments and thus, ethical approval was not required for this study. The nucleotide sequence of the envelope protein (E-protein) of the isolated virus (GenBank accession number AB690858) was compared to selected sequences of DENV-3. The isolated DENV-3 strain from Benin belonged to DENV-3,

genotype III (Figure 1) and had the following characteristics: an E-protein sequence similarity of 99% to the DENV-3 D3/Hu/Côte d’Ivoire/NIID48/2008 strain, 99% to a DENV-3 strain isolated in Senegal in 2009, and 98% to a DENV-3 D3/Hu/Tanzania/NIID08/2010 strain isolated in Tanzania in 2010 (GenBank accession numbers: AB447989, GU189386, and AB549332, respectively). Sporadic cases or outbreaks of DENV infection have been reported in 34 countries in the African region. It is estimated Thymidylate synthase that 2.4% of global dengue hemorrhagic fever (DHF) cases (100,000 cases) and up to 1 million cases of DF may occur in Africa.[2] Among travel-associated dengue cases in travelers returning to Europe, 2 to 8% had visited Africa.[2, 5] In comparison, most of

the travelers returning to Europe with dengue had traveled to Asia (54–61%) and Latin America (25–31%). Febrile illness was, however, more frequently reported in 41% of travelers to sub-Saharan Africa (2,559 patients) as compared to other regions (Southeast Asia, 33%, 1,218 patients; Caribbean and Central and South America, 18%, 1,044 patients).[9] Although dengue is frequently reported in travelers to Southeast Asia and South America as compared to Africa, the disease may be underreported in Africa due to limited awareness of the disease, and, limited availability of diagnostic tests and routine surveillance system.[2] Imported cases of DENV type-3 infection from West Africa have been previously reported in European travelers.[2-6] The first possibility of DENV circulation in Benin was suggested by a seroprevalence study conducted in asymptomatic Germans working overseas from 1987 to 1993.

, 2004) and

the novel-sound P3a has been found to be enlarged in highly distractible children such as those with attention deficit Dapagliflozin concentration hyperactivity disorder (van Mourik et al., 2007) and major depression (Lepistö et al., 2004). In sum, a large P3a to subtle deviants appears to be associated with highly accurate auditory discrimination, whereas high-amplitude P3as to novel sounds may be indicative of heightened distractibility. The P3a responses elicited by novel sounds vs. more subtle deviant tones might also display distinct developmental trajectories. For frequency deviants, an age-related increase in P3a amplitude has been reported (Wetzel & Schröger, 2007a). Admittedly, very few studies have specifically examined the development of the deviant elicited P3a. By visual inspection of the figures,

a few studies (Gomot et al., 2000; Shafer et al., 2000; Horváth et al., 2009a) appear to support an age-related increase in the deviant-tone P3a but unfortunately these studies did not statistically examine the age differences in this response. In contrast, the novel-sound P3a seems to decrease (over the frontal electrodes) between preschool age and adulthood Roxadustat supplier (Määttä et al., 2005; Wetzel & Schröger, 2007b; Wetzel et al., 2011), which might be related the maturation of the frontal cortex. Therefore, the enlarged P3as to deviants and reduced P3as to novel sounds found in the children from more musically active homes could be speculated to reflect more mature response morphology. With regard to the novel-sound P3a, the correlation was specific to parental singing, whereas the correlation between this response and the overall musical activities at home score did not reach significance. This result indicates that, in particular, listening to informal musical performances (as opposed to more active musical play) is associated with

reduced distractibility. Parental singing is highly effective in maintaining the attention of young infants (Trehub, 2009). In fact, singing by the father might be especially engaging for infants as indicated by behavioural Abiraterone concentration measures of visual attention during listening to paternal vs. maternal singing (O’Neill et al., 2001). Several authors have proposed that formal musical training might enhance executive functions (Moreno et al., 2011; Bialystok & DePape, 2009; Dege et al., 2011; however, see Schellenberg, 2011) such as selective attention (Trainor et al., 2009; Moreno et al., 2009). A recent longitudinal intervention study found support for these claims (Moreno et al., 2011). Our results indicate that even informal musical activity may also enhance functions related to auditory attention in childhood. Although a number of suggestions for the functional significance of the LDN have been put forward, the cognitive processes underlying this response remain to be disambiguated.

The first directs expression of the immediate upstream gene rpsO, and the second is positioned in the rpsO-pnp intergenic region (Portiers & Reginer, 1984). Irrespective of the transcriptional start site, the pnp mRNA is vulnerable to cleavage by endoribonuclease RNase III at positions

within 75 nucleotides upstream the pnp ORF, which in turn initiates degradation of the pnp mRNA by PNPase itself (Portier et al., 1987). Upon a cold shock, the pnp mRNA becomes stabilized allowing enhanced expression of PNPase (Beran & Simons, 2001). In enterobacteria, pnp is followed by nlpI (Blattner et al., 1997; McClelland et al., 2001; Nie et al., 2006). For E. coli, NlpI has been shown to be a lipoprotein (Ohara et al., 1999). We recently demonstrated that PNPase and NlpI posed opposing effect on biofilm formation in S. Typhimurium Ku 0059436 at decreased growth temperature (Rouf et al., 2011). Experiments that followed here demonstrate that mutational inactivation of pnp in S. Typhimurium results in an expected restricted growth at 15 °C. In addition, the experiments showed that pnp transcripts continued into nlpI and that nonpolar pnp mutations increased nlpI expression. Although S. Typhimurium pnp and nlpI are separated

Copanlisib purchase by 109 base pairs, the promoter prediction software bprom (www.Softberry.com) failed to define any tentative nlpI promoter within this intergenic region (data not shown). Combined with the gene expression analysis, this strongly suggests that pnp and nlpI form an operon and implies that nlpI is subject to the same post-translational regulation of pnp. However, we cannot formally exclude potential nlpI promoters within pnp. The co-transcription of pnp and nlpI led us to detail whether, and to what extent, NlpI contributed to cold acclimatization. The data presented in this study demonstrate that nlpI does indeed functionally act as a cold shock gene in concert with, but independently of, pnp. Evidence to support includes the observation that two of the the three pnp mutants applied in this study had enhanced expression of nlpI, whilst the third had unaffected nlpI mRNA levels compared

to the wild type, yet all three mutants showed a very similar defect for growth at 15 °C. In addition, a pnp–nlpI double mutant had more restricted growth at 15 °C compared to either single mutant, whilst cloned pnp and nlpI enhanced the replication of all the respective mutants at 15 °C (Figs 4b and 5). The nlpI gene is adjacent to csdA/deaD in the genomes of enterobacteria (Blattner et al., 1997; McClelland et al., 2001; Nie et al., 2006). The csdA gene encodes for an alternative RNA helicase that in E. coli also contributes to cold acclimatization (Turner et al., 2007). In S. Typhimurium, the homologue for csdA is defined as deaD. Deleting deaD in S. Typhimurium resulted in a cold-sensitive growth phenotype. However, we could not trans-complement the cold-restricted growth of the deaD mutant phenotype with either pnp or nlpI.

The number of visible viral lesions was

counted and the diameter and area of lesions were measured 6 days after inoculation. For each treatment, six plants were used and each experiment find more was repeated three times. The production of superoxide anion radical (O2−) and peroxide (H2O2) in the leaves of the 8–10-leaf stage tobacco plants treated with Trichokonins or control solution were examined using the procedure of Fitzgerald et al. (2004). For detection of systemic responses, seedlings were cultured in MS-medium containing 100 nM Trichokonins or control solution for 4 days, after which the top leaf was harvested. For detection of local responses, Trichokonins (100 nM) or 2 μL control solution were placed on the adaxial surface beta-catenin inhibitor of scratched leaves. Leaves were harvested and analyzed immediately. The leaves treated with Trichokonins or control solution were vacuum-infiltrated with nitrotetrazolium blue chloride (NBT) or 3,3-diaminobenzidine (DAB), incubated overnight at 28 °C, fixed and cleared in alcoholic lactophenol solution and examined for the formation of precipitates. Microscopic analysis was performed using an Olympus Stereoscope SZX-9 (Olympus America Inc., Melville, NY)

at × 40 magnification. To test autofluorescence, 2 μL of 100 nM Trichokonins or 2 μL control solution were placed on the adaxial surface of scratched leaves. After 24 h incubation at 25±1 °C, the autofluorescence in the leaves was assessed (Fitzgerald et al., 2004). Microscopic analysis was performed using Olympus BX-51 fluorescent microscope (Olympus America Inc.) at × 200 Amobarbital magnification. The excitation wavelengths were 470–490 nm and the emission wavelengths were 510–550 nm. Each tobacco plant at the 8–10-leaf stage was sprayed with 1 mL of 100 nM Trichokonins or 1 mL control solution. After 0, 1, 2, 3, 4, 5 or 6 days, the leaves of tobacco plants were harvested, ground to a fine powder in liquid

nitrogen and stored at −80 °C until analysis. Phenylalanine Ammonia-Lyase (PAL, E.C.4.3.1.5) activity was determined as described by González-Aguilar et al. (2004). Peroxidases (POD, E.C.1.11.1.7) activity was determined as described by Rathmell & Sequeira (1974). Polyphenol oxidases (PPO, E.C. 1.14.18.1) activity was assayed using Flurkey’s method (Flurkey, 1985). For each treatment, three tobacco plants were used and each experiment was repeated three times. RT-PCR analysis was conducted to determine the expression of selected defense-related genes in Trichokonins-treated tobacco plants. Each tobacco plant at the 8–10-leaf stage was sprayed with 1 mL of 100 nM Trichokonins. Total RNA was extracted from treated tobacco leaves after 0, 1, 2, 4, 6, 9, 12, 24 or 48 h treatment. The quality of extracted RNA was tested by 1.0% agarose electrophoresis. The transcription levels of genes were detected by RT-PCR using a One Step RNA PCR Kit (TaKaRa, Japan). RT-PCR products were loaded on 1.