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2.2. EBC Was Attractive to Non-Viruliferous N. Virescens, while 2-Heptanol Was Repellent to Viruliferous N. Virescens

Behavioral assays revealed that 2-heptanol was not attractive or repellent to non-viruliferous N. virescens at any of the three dosages tested (C, 0.01 μg μL−1, χ2 = 4.832, P = 0.185, n = 38; 0.1 μg μL−1, χ2 = 0.270, P = 0.966, n = 40; 1 μg μL−1, χ2 = 1.215, P = 0.749, n = 41). However, 2-heptanol was significantly repellent to viruliferous N. virescens at the three dosages tested (D, 0.01 μg μL−1, χ2 = 58.304, p < 0.001, n = 36; 0.1 μg μL−1, χ2 = 44.367, p < 0.001, n = 36; 1 μg μL−1, χ2 = 82.395, p < 0.001, n = 37). The walking paths of N. virescens are shown in (2-heptanol, 0.1 μg μL−1).

2.2. EBC Was Attractive to Non-Viruliferous N. Virescens, while 2-Heptanol Was Repellent to Viruliferous N. Virescens

Behavioral assays revealed that 2-heptanol was not attractive or repellent to non-viruliferous N. virescens at any of the three dosages tested (C, 0.01 μg μL−1, χ2 = 4.832, P = 0.185, n = 38; 0.1 μg μL−1, χ2 = 0.270, P = 0.966, n = 40; 1 μg μL−1, χ2 = 1.215, P = 0.749, n = 41). However, 2-heptanol was significantly repellent to viruliferous N. virescens at the three dosages tested (D, 0.01 μg μL−1, χ2 = 58.304, p < 0.001, n = 36; 0.1 μg μL−1, χ2 = 44.367, p < 0.001, n = 36; 1 μg μL−1, χ2 = 82.395, p < 0.001, n = 37). The walking paths of N. virescens are shown in (2-heptanol, 0.1 μg μL−1).

Background

The human gut harbors a diverse community of microorganisms which serve numerous important functions for the host wellbeing. Functional foods are commonly used to modulate the composition of the gut microbiota contributing to the maintenance of the host health or prevention of disease. In the present study, we characterized the impact of one month intake of a synbiotic food, containing fructooligosaccharides and the probiotic strains Lactobacillus helveticus Bar13 and Bifidobacterium longum Bar33, on the gut microbiota composition and metabolic profiles of 20 healthy subjects.

Results

The synbiotic food did not modify the overall structure of the gut microbiome, as indicated by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The ability of the probiotic L. helveticus and B. longum strains to pass through the gastrointestinal tract was hypothesized on the basis of real-time PCR data. In spite of a stable microbiota, the intake of the synbiotic food resulted in a shift of the fecal metabolic profiles, highlighted by the Gas Chromatography Mass Spectrometry Solid Phase Micro-Extraction (GC-MS/SPME) analysis. The extent of short chain fatty acids (SCFA), ketones, carbon disulfide and methyl acetate was significantly affected by the synbiotic food consumption. Furthermore, the Canonical discriminant Analysis of Principal coordinates (CAP) of GC-MS/SPME profiles allowed a separation of the stool samples recovered before and after the consumption of the functional food.

Effects of the synbiotic food on composition of the gut microbiota

PCR-DGGE analysis with universal primers targeting the V2-V3 region of the 16S rRNA gene was used to monitor the impact of the synbiotic food intake on the predominant bacterial population (Figure ). Population fingerprint profiles were compared and numerically analyzed by FPQuest Software. DGGE band profiles (mean of bands: 15.3) were stable for each subject over a month of feeding with the functional food. Only a slight difference in band richness was found between the time points of the study (T0, mean of bands: 15.8; T1, mean of bands: 14.8). DGGE bands were subjected to Mann-Whitney U-test in order to search for significant differences in the intensities between T0 and T1. No band showed a significant variation, indicating that the consumption of the synbiotic food did not alter the concentration of any major species of intestinal microbiota. Pearson correlation was used to calculate the similarity index (SI) between DGGE band profiles related to the time points T0 and T1 for each healthy volunteer (Table ). The high median value of SI (67.1%) revealed that the dominant bacterial composition remained constant over the treatment. Only 3 subjects presented SIs lower than 50% (subjects 8, 12 and 20). No subject showed significant variations between DGGE band profiles related to T0 and T1, as evaluated using the Pearson correlation analysis (P > 0.05).

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

To understand the cause of the cell wall stress in the rgc1/2Δ mutant, we conducted a dosage suppressor screen for high-copy number plasmids that conferred growth at 37°C. A single class of strong suppressor was identified as the FPS1 gene (). FPS1 encodes an aquaglyceroporin that is the major facilitator of glycerol uptake and efflux in yeast ,. This plasma membrane channel protein also mediates uptake of toxic metalloids, such as arsenite and antimonite ,. One interpretation of the suppression result is that the rgc1/2Δ mutant experiences abnormally high turgor pressure from accumulation of glycerol, which yeast cells use as a compatible solute for osmoregulation. Measurement of intracellular glycerol concentration confirmed that the rgc1/2Δ mutant has a 5.9-fold higher glycerol level than wild-type cells under normal growth conditions, a value that is approximately half that of an fps1Δ mutant and approximately equal to that of wild-type cells exposed to hyper-osmotic shock (). To determine if excess intracellular glycerol is responsible for the phenotypic defects of this mutant, we blocked glycerol biosynthesis at the first committed and rate limiting step, glycerol-3-phosphate dehydrogenase (GPD) –. GPD is encoded by the paralogous genes GPD1 and GPD2. Deletion of either GPD1 or GPD2 alone did not suppress the lysis defect of the rgc1/2Δ mutant, but blocking glycerol biosynthesis completely by deletion of both GPD1 and GPD2 allowed growth at 37°C (), confirming that glycerol accumulation is responsible for the cell lysis defect. This also provides an explanation for the fortified cell wall of the rgc1/2Δ mutant as a response to the stress of abnormally high turgor pressure. Consistent with this interpretation, the gpd1/2Δ mutations relieved the cell wall stress signaling observed in the rgc1/2Δ mutant (). Finally, the gpd1/2Δ mutations relieved the caspofungin sensitivity of the rgc1/2Δ mutant ().

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

We considered two possible explanations for the hyper-accumulation of glycerol in the rgc1/2Δ mutant – the mutant either produces excess glycerol, or it is impaired for glycerol efflux through Fps1. These hypotheses generate different predictions for the impact of the rgc1/2Δ mutations on the phenotype of an fps1Δ mutant. If the rgc1/2Δ mutant produces excess glycerol, this should exacerbate the lysis defect of an fps1Δ mutant, which is blocked for glycerol efflux. By contrast, if the rgc1/2Δ mutant is blocked for glycerol efflux through Fps1, loss of the glycerol channel should not result in an additive defect. shows that an fps1Δ mutant displays a temperature-sensitive growth defect that is slightly more severe than that of the rgc1/2Δ mutant, with a semi-permissive growth temperature of 34.5°C. The fps1Δ mutant growth defect at elevated temperature is also the result of cell lysis (data not shown). Significantly, the rgc1/2Δ fps1Δ triple mutant behaves identically to the fps1Δ mutant (), supporting the hypothesis that the rgc1/2Δ mutant is impaired for glycerol efflux through Fps1. To test this directly, we measured export of glycerol from cells exposed to a hypo-osmotic shock, a condition that would induce glycerol efflux through Fps1. We found that glycerol was released from wild-type cells, but not from the rgc1/2Δ mutant or the fps1Δ mutant (), supporting the conclusion that Rgc1 and Rgc2 regulate glycerol efflux through Fps1. The varied initial content of 14C-labeled glycerol among these mutants is a consequence of differential glycerol loading, reflecting the importance of Fps1 for glycerol influx as well as efflux. Finally, strong overexpression of RGC2 failed to suppress the temperature-sensitivity of the fps1Δ mutant (data not shown), thus establishing an epistatic relationship that places RGC1 and RGC2 above FPS1 in a common pathway for glycerol efflux.

Rgc1 and Rgc2 serve a redundant role in the regulation of glycerol efflux through the Fps1 glycerol channel

To determine the cause of the increased steady-state level of Fps1 in the rgc1/2Δ mutant, we conducted a test of Fps1 stability. Fps1 levels were followed in cells in which FPS1 transcription was shut down with the simultaneous inhibition of protein synthesis. We found that Fps1 was stabilized in the rgc1/2Δ mutant relative to wild-type cells (). Therefore, we conclude that increased intracellular glycerol in the rgc1/2Δ mutant, which is caused by a deficiency in Fps1 function, induces an increase in the level of weakly functional Fps1 through protein stabilization.

4. Curcumin Safety and Toxicity

In the systematic studies funded by the Prevention Division of the US National Cancer Institute (NCI) conducted in rats, dogs, or monkeys and at oral doses of curcumin up to 3,500 mg/kg-BW for up to 90 days, no adverse effects were observed []. In a preclinical study involving the administration of 2% dietary curcumin (approx. 1,200 mg/kg-BW) to rats for 14 days [] or in a study of 0.2% dietary curcumin (approx. 300 mg/kg-BW) administered to mice for 14 weeks, no toxicity was observed []. Furthermore, a reproductive toxicity study with the oral curcumin administration of up to 1,000 mg/kg-BW daily, no toxicity was observed in two successive generations in rats [].

4. Curcumin Safety and Toxicity

In a study performed in India, daily administration of 1,200–2,100 mg of oral curcumin to patients with rheumatoid arthritis for 2–6 weeks did not cause any toxicity []. In another study of high-dose oral curcumin by Cheng et al. in Taiwan, administration of 500, 1,000, 2,000, 4,000, and 8,000 mg of curcumin daily for three months to patients with preinvasive malignant or high risk premalignant conditions, no noticeable adverse effects were detected []. Lao et al. studied safety of curcumin in healthy volunteers using curcumin capsules (containing 75% curcumin, 23% demethoxycurcumin and 2% bisdemethoxycurcumin) with single escalating doses from 500 mg to 12,000 mg. Among 24 enrolled healthy subjects, seven developed adverse effects, including diarrhea, headaches, rashes and yellowish stools. All toxicities observed were of grade 1 and not related to the doses. In this study, the maximum tolerated dose could not be determined because more than 12,000 mg of curcumin is regarded as too bulky [].

4. Curcumin Safety and Toxicity

In a study performed in India, daily administration of 1,200–2,100 mg of oral curcumin to patients with rheumatoid arthritis for 2–6 weeks did not cause any toxicity []. In another study of high-dose oral curcumin by Cheng et al. in Taiwan, administration of 500, 1,000, 2,000, 4,000, and 8,000 mg of curcumin daily for three months to patients with preinvasive malignant or high risk premalignant conditions, no noticeable adverse effects were detected []. Lao et al. studied safety of curcumin in healthy volunteers using curcumin capsules (containing 75% curcumin, 23% demethoxycurcumin and 2% bisdemethoxycurcumin) with single escalating doses from 500 mg to 12,000 mg. Among 24 enrolled healthy subjects, seven developed adverse effects, including diarrhea, headaches, rashes and yellowish stools. All toxicities observed were of grade 1 and not related to the doses. In this study, the maximum tolerated dose could not be determined because more than 12,000 mg of curcumin is regarded as too bulky [].

4. Curcumin Safety and Toxicity

In a phase I clinical trial on oral curcumin in patients with advanced colorectal cancer in which the US NCI criteria were used to assess potential toxicity, curcumin was well tolerated at all dose levels up to 3,600 mg daily for up to four months []. Adverse effects related to curcumin consumption reported by patients in these studies were mainly gastrointestinal (nausea and diarrhoea). Two abnormalities were detected in blood tests in this trial, both possibly related to treatment: an increase in serum alkaline phosphatase level was observed in four patients (two NCI grade 1, and two grade 2); and three other patients had serum lactate dehydrogenase increases to >1.5 times the upper limit of normal. It is not clear whether these abnormal blood test results were related to the activity of the malignant disease in these patients or to treatment toxicity.

5.1. Animal Model Pharmacokinetics

Due to its poor systemic bioavailability after oral administration, many research groups have focused on ways to improve its bioavailability. Co-administration of oral curcumin with piperine, an alkaloid found in black pepper (Piper nigrum) and long pepper (Piper longa), appeared to increase serum concentrations of curcumin in rodents. In a study using high doses of oral curcumin (2,000 mg/kg) in rats, the investigators found that co-administration of piperine increased systemic bioavailability by as much as 154% []. The mechanism of this effect has not been elucidated yet.

Abstract

Compartmentalized redox faults are common to ageing diseases. Dietary constituents are catabolized to NAD(H) donating electrons producing proton-based bioenergy in coevolved, cross-species and cross-organ networks. Nicotinamide and NAD deficiency from poor diet or high expenditure causes pellagra, an ageing and dementing disorder with lost robustness to infection and stress. Nicotinamide and stress induce Nicotinamide-N-methyltransferase (NNMT) improving choline retention but consume methyl groups. High NNMT activity is linked to Parkinson's, cancers, and diseases of affluence. Optimising nicotinamide and choline/methyl group availability is important for brain development and increased during our evolution raising metabolic and methylome ceilings through dietary/metabolic symbiotic means but strict energy constraints remain and life-history tradeoffs are the rule. An optimal energy, NAD and methyl group supply, avoiding hypo and hyper-vitaminoses nicotinamide and choline, is important to healthy ageing and avoids utilising double-edged symbionts or uncontrolled autophagy or reversions to fermentation reactions in inflammatory and cancerous tissue that all redistribute NAD(P)(H), but incur high allostatic costs.

2. Pellagra

An NAD(H) deficiency state that illustrates the complexity of these coevolved energy networks was observed during the largely forgotten epidemics of pellagra caused by economically determined suboptimal foraging of nicotinamide and tryptophan (tryptophan is converted to NAD and serotonin) and dietary sources of methyl-groups. Such a dietary deficiency may still be important in sections of modern populations and intracellular and microenvironmental deficiency states can also occur from high NAD expenditure with NAD(H) ratios also affected by any disorder that causes hypoxia or hypoglycemia [, ].

2. Results and Discussion

2-Octanol was successfully oxidized in nearly quantitative yield both by the composite and 30% aqueous H2O2 (). The application of UHP as an oxidant lowers the yield of the reaction to 69%. Moreover, when the reaction temperature was decreased to 70 °C, UHP exhibited low reactivity manifested only 6% yield of 2-octanone with comparison to 64% and 56% for aqueous H2O2 and the xerogel, respectively. Oxidation under microwave conditions gave very good results only when 30% H2O2 was applied.

Abstract

The present study deals with the preparation of polyurethane (PU) films impregnated with a nonsteroidal anti-inflammatory drug, tolfenamic acid (TA). Solvent evaporation technique has been employed for the preparation of TA-PU films in two different ratios of 1 : 2 and 1 : 5 in Tetrahydrofuran (THF) or THF-ethanol mixtures. The prepared films were characterized using X-Ray Diffraction (XRD), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and release studies. The results indicate transformation of crystalline TA to its amorphous form. The degree of crystallinity changes both by increasing the polymer concentration and solvent used for the film preparations. The release profiles of TA were also found to be affected, showing a decrease from approximately 50% to 25% from 1 : 2 to 1 : 5 ratios, respectively.

3.1. Appearance of TA-PU Films

Equal volumes of the solutions were used in the preparation of all films. However, TA-PU films prepared by two different methods showed some differences in their appearance. The films prepared by method I (both drug and polymer in THF) were found to be more transparent with smooth surface whereas the films prepared by method II (drug in ethanol and polymer in THF) appeared opaque with slightly rough surface. No signs of recrystallization or changes in appearance took place in all the films during storage for up to 3 months in a desiccator.

3.2.1. XRD Studies

The pure crystalline TA shows characteristic diffraction peaks at 2θ values of 5.21°, 11.58°, 15.72°, 18.70°, 19.77°, 24.90°, 25.32°, 25.90°, and 26.81°, respectively (). In case of PU, a hallow diffraction pattern with no sharp peaks has been observed in the pure as well as in the control films thus indicating its amorphous nature. A typical diffraction pattern has been presented in . The diffraction patterns of the 1 : 2 TA-PU films prepared by method I indicated a marked decrease in the peaks intensity of the crystalline TA and show very small peaks at around 15.72–18.70° and 24.90–26.81° whereas the remaining peaks of the drug have not been detected (). The diffraction pattern of 1 : 5 TA-PU films also shows similar type of result with comparatively less intense peaks in the same region (). This decrease in peak intensity clearly indicates that majority of drug has been transformed to the amorphous form.

3.2.1. XRD Studies

The films prepared using ethanol for drug solubilisation (method II) showed similar type of diffraction patterns for 1 : 2 and 1 : 5 ratios, respectively, but the intensity of the peaks is comparatively higher to the films prepared in THF only (method I) (Figures and ). Therefore, method I could be considered as more effective comparatively in converting crystalline TA to its amorphous form. Similarly, the difference in the diffraction pattern of the films indicates that the solvent also plays an important role in modifying the solid state properties of the drug in addition to the technique used and the nature of the polymer. THF evaporates more rapidly as compared to ethanol-THF mixture [] and therefore the use of THF alone has resulted in greater amorphicity of the drug. The samples were further subjected to analysis by DSC, FTIR, and SEM techniques in order to confirm the results obtained by XRD.

Abstract

Snakebites are a major Collective Health problem worldwide. In Brazil, Bothrops jararaca snake venom (BjV) evokes hemostatic disturbances, bleeding manifestations, and redox status imbalance. Specific antivenom therapy, although efficacious to revert most snakebite-induced manifestations, is incapable of treating secondary manifestations, such as oxidative/nitrosative stress. Searching for new complementary therapies that could attenuate physiological derangements triggered by envenomation, we elected to test quercetin-3-rutinoside (rutin) by its potential as both a potent antioxidant and a hemostasis modulatory compound. The activity of rutin was evaluated both on the biological activities of crude BjV in vitro, and in vivo by the ability of rutin (14.4 mg/kg b.w.) to modulate hematological, hemostatic and redox status markers altered by BjV injection (1.6 mg/kg b.w., s.c.) in mice. In vitro, rutin failed to inhibit BjV-induced platelet aggregation and biological activities of major BjV enzymes (metalloproteinases, phospholipases A2, serine proteases, and L-amino acid oxidases). On the other hand, rutin attenuated local hemorrhage, and the increase in reactive species, prevented the fall in RBC counts and fibrinogen levels, diminished tail bleeding and shortened prothrombin time (PT) evoked by envenomation. Furthermore, rutin reduced tissue factor (TF) activity and altered the protein expression of TF in liver, lungs, heart and skin. In conclusion, the disturbances in redox status and hemostatic system induced by B. jararaca envenomation were modulated by rutin, suggesting it has a great potential to be used as an ancillary therapeutic agent for snakebites.

Ethics statement

Male Swiss mice, weighing 30–35 g, were obtained from the Animal Facility of Instituto Butantan, and were maintained with free access to food and water. The experimental procedures involving human donors, rats, and mice were in accordance with National Guidelines, and were approved, respectively, by the National Human Research Ethics Committee (Plataforma Brasil, CAAE: 51368615.5.0000.0065), and the Institutional Animal Care and Use Committee from Instituto Butantan (CEUAIB 4388061115) and the Faculdade de Medicina, Universidade de São Paulo (protocol 188/15). All procedures involving animals were in accordance with the National Guidelines of Conselho Nacional de Controle de Experimentação Animal (CONCEA) [].

Venom and rutin solutions

BjV was dissolved in sterile saline (0.8 mg/mL); rutin (7.2 mg/mL) was dissolved in a solution containing 1 vol. of propylene glycol and 1 vol. of saline. The solutions were prepared immediately before use. The dose of BjV (1.6 mg/kg b.w., s.c. route) was selected based on previous tests to determine a dose that evoked hemostatic disturbances in mice similar to those observed in rats [, ] and humans []. The dose of rutin (14.4 mg/kg b.w, s.c. route) was established based on previous experiments, showing that this dose was effective to modulate redox status and hemostatic parameters.

Experimental groups and procedures

Animals were randomly allocated in four experimental groups (n = 6/group/time interval) () and received (4.0 mL/kg b.w., s.c.): saline (saline control, i.e., the negative control); rutin (rutin control, 14.4 mg/kg b.w. of rutin); BjV+saline (positive control, 1.6 mg/kg b.w. of BjV); and BjV+rutin (rutin treatment, 14.4 mg/kg b.w. of rutin and 1.6 mg/kg b.w. of BjV). The solutions were incubated at 37°C for 30 min before administration.

Experimental groups and procedures

At 3, 6 and 24 h after injection, animals (5–6 mice/group) were anesthetized with isoflurane (induction and maintenance at 2.5%), and blood and tissues were collected (). Blood was collected from the caudal vena cava into plastic syringes to obtain complete blood cell counts (CBC), blood smears and plasma. Blood was added to flasks containing the following anticoagulants: CTAD (75 mM trisodium citrate, 42 mM citric acid, 139 mM dextrose, 15 mM theophylline, 3.7 mM adenosine, 0.2 mM dipyridamole, and 2 μM imipramine), 3.2% trisodium citrate, or 269 mM Na2EDTA. Bothrops antivenom (1 vol. to 100 vol. of blood) was added to all flasks to avoid in vitro clotting of the blood induced by BjV. Fragments of liver, lungs, heart (50–100 mg) and skin (0.5–0.75 cm2 around the site of s.c. injection) were collected in flasks containing RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM Na2-EDTA, and a protease inhibitor cocktail, pH 7.5) or HBSA buffer (20 mM HEPES-NaOH buffer, pH 7.5, containing 100 mM NaCl, 0.02% NaN3 and 1 mg/mL bovine albumin), both containing Bothrops antivenom, and frozen at -80°C. Tissues were disrupted using FastPrep-24 (#6004–500, MP Biomedicals) for 60 s at 6.5 m/s, followed by 3 cycles of freezing in dry ice and thawing in a water bath at 37°C. Finally, samples were centrifuged at 13000 g for 10 min at 4°C, and supernatants were collected and frozen at -80°C. Protein concentration in the supernatants was determined by bicinchoninic acid assay []. As reference samples, pools of normal plasma and each organ (liver, lungs, heart and skin) were obtained and assayed on each determination.

Abstract

Conclusion: Incorporation of lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity.

Abstract

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

2.1. Q3R Protects Cultured Cells from CT

We hypothesized Q3R would block the cytopathic activity of CT through its inhibitory action on PDI. To test this prediction, cAMP levels were quantified from CHO cells incubated with CT for 2 h in the absence or presence of Q3R. As shown in A, Q3R-treated cells were protected from the cytopathic activity of CT. In contrast, Q3R did not inhibit the cAMP response generated from cells expressing an ER-localized CTA1 construct (B). Transfection with the pCDNA3.1/ssCTA1 plasmid results in co-translational insertion of CTA1 into the ER lumen, proteolytic cleavage of its N-terminal ER-targeting signal sequence (ss), and dislocation of CTA1 back into the cytosol where it elicits a cAMP response beginning 2 h post-transfection []. We found the cAMP response from Q3R-treated cells was 11% greater than the response from untreated cells that were also expressing CTA1. The basis for this mild sensitization remains to be determined, but the result clearly shows that Q3R does not block (i) the ER-to-cytosol export of free CTA1 or (ii) the enzymatic activity of cytosolic CTA1. The inhibitory effect of Q3R on CT intoxication thus occurs at a step preceding translocation of free CTA1 to the cytosol.

2.2. Q3R Does Not Prevent PDI from Binding or Reducing CTA1

Surface plasmon resonance (SPR) was used to examine the impact of Q3R on PDI binding to CTA1 (A). For this experiment, CTA1 was coupled to a SPR sensor. A baseline measurement corresponding to the mass of sensor-bound CTA1 was set at 0 refractive index units (RIU), and Q3R-treated PDI was then added to the perfusion buffer. The elevated signal following this injection indicated Q3R-treated PDI had bound to CTA1, thereby adding mass to the sensor surface and altering the RIU. The pattern of binding was similar to what was previously reported for untreated PDI []. Thus, Q3R did not disrupt the physical association between PDI and CTA1.

2.3. Q3R Disrupts PDI-Driven Disassembly of the CT Holotoxin

Our collective data indicated Q3R does not block PDI binding to CTA1, the PDI-induced shift in CTA1 protease sensitivity, or reduction of the CTA1/CTA2 disulfide bond by PDI. We next examined the effect of Q3R on disassembly of the CT holotoxin by PDI (). For this experiment, CT was appended to a GM1-coated SPR sensor slide. A baseline measurement corresponding to the mass of the captured holotoxin was then set at 0 RIU. Subsequent addition of PDI to the perfusion buffer resulted in an elevated RIU signal indicative of PDI binding to CT. In the absence of Q3R (A), the rise in RIU was followed by a rapid drop in signal to a point below the baseline value corresponding to the mass of the CT holotoxin. This drop occurred despite the continued presence of PDI in the perfusion buffer and was attributed to the PDI-mediated release of CTA1 from the sensor-bound CTA2/CTB5 complex. Both PDI and its displaced CTA1 binding partner would be removed from the sensor by the flow of the perfusion buffer, thus lowering the RIU below its initial baseline value. After removing PDI from the perfusion buffer, sequential antibody injections confirmed our interpretation: no signal was obtained from the PDI and CTA1 antibodies, whereas the CTB antibody produced a positive result. This result was consistent with previous reports that used SPR to monitor the PDI-driven disassembly of CT [,].

2.3. Q3R Disrupts PDI-Driven Disassembly of the CT Holotoxin

As with untreated PDI, injection of Q3R-treated PDI into the perfusion buffer produced an elevated RIU (B). However, no loss of signal occurred while Q3R-treated PDI was in the perfusion buffer. The RIU only dropped when Q3R-treated PDI was removed from the perfusion buffer, which indicated PDI had been released from the intact CT holotoxin. Yet the signal remained above the initial baseline measurement and never dropped below 0 RIU. It thus appeared that some PDI remained associated with the sensor-bound holotoxin for an extended time. This interpretation was confirmed by the positive signals generated from each sequential perfusion of anti-PDI, anti-CTA1, and anti-CTB antibodies over the sensor. Q3R-treated PDI could thus bind to the CT holotoxin but could not efficiently separate CTA1 from CTA2/CTB5.