, 2000). The amino acid sequences were retrieved from the Universal Protein Resource Knowledgebase (www.uniprot.org) or the Worldwide Protein Data Bank (www.pdb.org). Ts2 (Uniprot ID P68410) contains 62 amino acids, including 8 cysteine BMS-754807 in vitro residues ( Mansuelle et al., 1992). The peptide has a theoretical molar mass of 6998.0 Da and a pI of 7.70 (http://web.expasy.org/protparam/). Ts2 is a sodium channel inhibitor α-neurotoxin that

inhibits the rapid inactivation of NaV1.2, NaV1.3, NaV1.5, NaV1.6 and NaV1.7, but does not affect NaV1.4, NaV1.8 or DmNaV1 ( Cologna et al., 2012). The overall structure of the Ts2 model consists of three β-strands and one α-helix that are arranged in a triangular shape forming a cysteine-stabilized α-helix ⁄ β-sheet (CSαβ) motif ( Cologna et al., 2012). While Ts6, also called TsTX-IV ( Arantes et al., 1989), acts on K+ channels ( Coronas et al., 2003). Ts6 (Uniprot ID P59936) contains 40 amino acids, including 8 cysteine residues ( Pimenta et al., 2003).

The theoretical molar mass and pI of the peptide are 4514.2 Da and 8.50, respectively (http://web.expasy.org/protparam/). Ts6 is a potassium channel toxin alpha-KTx 12.1 that inhibits high conductance calcium-activated potassium channels ( Novello et al., 1999) and weakly inhibits Shaker B potassium channels ( Coronas et al., 2003). The structure of the peptide consists of an alpha-helix connected to a triple-stranded beta-sheet stabilized by four disulfide bonds ( Oyama et al., 2005). Selleckchem Obeticholic Acid Our group has previously demonstrated that following stimulation with Ts1, Ts2 or Ts6, the in vitro release Unoprostone of inflammatory mediators by peritoneal macrophages (J774.1) is independent of the action of these toxins on ion channels ( Zoccal et al., 2011). Several reports demonstrated that cytokines are increased after envenomation. Magalhães et al. (1999) observed an increase of interleukins (IL)-1α, IL-6, Interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the serum of patients who were stung by T. serrulatus. Furthermore, the increased

levels of GM-CSF in severe envenomation might be involved in neutrophilia induced by T. serrulatus venom ( Magalhães et al., 1999). The synthesis of anti-inflammatory cytokines, such as IL-10, was also observed in the plasma of patients with both moderate and severe cases of envenomation ( Fukuhara et al., 2003; Petricevich, 2010). Scorpion venoms can stimulate the immune-neuroendocrine axis by inducing the release of catecholamines, corticosteroids, bradykinin ( Chaudry et al., 1989; Sofer et al., 1996; Magalhães et al., 1999; Pessini et al., 2003) and eicosanoids mediators, such as prostaglandin (PG)E2, lipoxin A2 (LXA2) and leukotriene (LT)B4 ( Nascimento et al., 2005; Teixeira et al., 1997).

However, the benefits of rotavirus

vaccination against severe diarrhea and death from rotavirus infection far exceed the miniscule risk of intussusceptions. It urges the manufacturers to actively monitor the risk of intussusceptions as the usage of these vaccines is bound to go up. This will also require strengthening of AEFI surveillance in the country. Information about the possible risk of intussusceptions associated with rotavirus vaccination needs to be communicated clearly to the national decision-makers, healthcare providers, and parents. The committee also stresses the need of strictly adhering to the set upper age limits of these vaccines, i.e. the first dose of either RV1 or RV5 should be administered between Integrase inhibitor the ages of 6 weeks and 14 weeks and 6 days, and that the maximum age for administering the last dose of INCB024360 supplier either vaccine should be 32 weeks25 of these vaccines while prescribing them in office practice. The committee has recommended inclusion of the history of intussusception in the past as an absolute

contraindication for rotavirus vaccine (RV1 and RV5) administration. The committee studied the recent data on PCV13 and PCV10. The committee also reviewed the reports of PCV13 studies done worldwide on immune responses (IgG – GMC, OPA – GMT) and boostability for the serotype 3 capsular antigen,26 and the immune responses following post-primary and post-booster series against serotype 19A infections, with PCV10 and PCV13.27 and 28 It has reviewed the interim data of COMPAS trial done in three Latin American countries with PCV1029 and effectiveness of PCV10 in Brazil.30

The committee also reviewed available data on the efficacy of the new serotypes in the PCV13. In England and Wales,31 vaccine effectiveness (VE) for the new serotypes for 2 doses under a year was 78% (95% CI: −18 to 96%) and 77% (CI: 38–91%) for Isotretinoin one dose over a year. VE for 7F and 19A was 76% (CI: 21–93%) and 70% (CI: 10–90%), respectively for ≥ one dose, for serotypes 1 and 3 was 62% and 66%, respectively although confidence intervals spanned zero. IPD due to PCV13-only serotypes halved in children under 2 years in the study period.31 The committee believes that the direct protection rendered by the serotype included in a vaccine formulation is definitely superior to any cross protection offered by the unrelated serotypes even of the same group in a PCV formulation. However, the committee still not convinced about the clinical efficacy of serotype 3 contained in PCV13 despite multiple studies showing good functional immune responses after the infant series29 and reasonably good effectiveness.31 There has been no consistent PCV13 impact on serotype 3 IPD or carriage reported so far.

Since caspase-3 activation is a central feature of apoptotic cells, further investigation of the pathways involving CdTe-QD-induced apoptosis was done. Our results showed that CdTe-QDs caused an increase in Fas level and in caspase-8 activity indicating that CdTe-QDs cause apoptosis in HepG2 cells via extrinsic pathways. Our findings align with the previous study by Choi et al. who reported that CdTe-QD treatment to human neuroblastoma cells caused apoptosis associated with increased Fas expression ( Choi et al., 2007). Our results also showed that exposure to CdTe-QDs caused effects on anti-apoptotic protein Bcl2 and pro-apoptotic protein Bax levels, which are biomarkers for the intrinsic pathway ( Dejean

et al., 2006). Bcl2 is a key inhibitor of apoptosis since its over-expression blocks translocation of cytochrome c from mitochondria into cytosol, SCR7 nmr thus preventing cells from undergoing apoptosis ( Yang et al., 1997).

Conversely, over-expression of Bax and its translocation to mitochondria has been shown to promote the release of cytochrome c into cytosol, leading to activation of effector caspases and subsequently to apoptosis ( Finucane et al., 1999). Our results showed CdTe-QDs caused a decrease in Bcl2 and an increase in mitochondrial Bax levels, suggesting intrinsic apoptotic pathway induction. The release of cytochrome c from mitochondria to cytosol confirmed the effects on cellular Bcl2 protein members. In addition, the present study also revealed that CdTe-QDs activated MAPK signaling pathways as indicated selleckchem by increases in phosphorylation levels of JNK and p38. These results are supported by a previous study reporting that activation of JNK and p38 caused phosphorylation and translocation of Bax into mitochondria to cause apoptosis via intrinsic pathway ( Kim et al., 2006). The present study also adds to the findings from the previous studies by Lu et al. (2006) who showed that activation of JNK was required for CdSe-QDs induced apoptosis in human osteoblast cells. Similarly, Chan et al. (2006) reported that CdSe-QDs induced apoptosis in human

neuroblasma cells via intrinsic (mitochondrial) pathway involving JNK activation. However, contrary to the report from Chan and colleagues who showed that their test C-X-C chemokine receptor type 7 (CXCR-7) QDs caused a decrease in Erk level resulting in inhibition of Ras to Erk survival signaling ( Chan et al., 2006), the present study observed an increase in Erk1/2. The reason for the difference between these findings might be due to differences in the test cell lines, as suggested in the existing literature that Cd-induced Erk activation is cell type-dependent ( Martin and Pognonec, 2010). Cadmium has also been shown to cause apoptosis in vitro and in vivo and the apoptosis induced by cadmium is suggested to arise from the effects of ROS generated by the metal ( Hamada et al., 1997 and Oh and Lim, 2006).

Tribl et al. carried out the first proteomic profile of intact neuromelanin (NM) granules enriched from control human SN using density gradient centrifugation [199]. Seventy-two proteins were identified, of which many were closely linked to lysosome-related organelles [199]. Of note, the protocol has been recently improved to allow the combined enrichment of neuromelanin Selleck MAPK Inhibitor Library and synaptosomal fractions using far less starting material (<0.15 g) [234]. This important development may

allow collecting a sufficient amount of NM from PD patient nigral tissues, which are severely depleted in NM- containing cells. A link between NM and PD pathogenesis was hypothesized as NM-containing neurons seem to be more vulnerable in PD [235]. Moreover, NM interacts with iron, which is known to accumulate in the parkinsonian SN. Recently, a targeted proteomic approach revealed that l-ferritin was an NM granule component, providing new clues on iron storage mechanisms in the NM-containing neurons [236]. These investigations provided insights JQ1 into NM composition, mechanisms and function, which are still poorly characterized, and may help to understand iron- driven degeneration of the SN in PD. To gain more insights into

the disease pathogenesis, quantitative proteomic data may allow the complex proteome alterations occurring in the brains of PD versus control patients to be disentangled. 2-DE studies of human brain tissues targeting the SN were Dipeptidyl peptidase conducted, highlighting several abnormalities in the proteome of PD patients [152], [153] and [192]. For example, our group was able to identify CNDP2 or VPS29 overexpression in PD. Using a shotgun approach combined to ICAT, others found 119 proteins exhibit changes in their relative expression in mitochondrial fractions obtained from the SNpc of PD cases compared to controls [196]. Of these, mortalin decrease in PD was confirmed using a cellular PD model and functional biology experiments suggested a major role

for mortalin in PD neurotoxicity through mechanisms that may involve oxidative stress, mitochondrial and proteasomal dysfunction [196]. Taking advantage of the sixplex TMT tagging technology to compare the nigral proteome of PD patients (n = 3) versus controls (n = 3), our group observed significant expression level changes in 204 proteins. PD-relevant candidates were further characterized including nebulette, whose overexpression might be associated to neurodegeneration in PD through mechanisms that may involve disruption of cytoskeletal dynamics [232]. A few proteomic comparative studies have focused on post-mortem cortical tissues. Two studies using iTRAQ labeling to profile frontal cortex samples of PD patients at different stage of the disease and control cases, suggested a potential association of respectively mortalin and glutathione-S transferase Pi (GSTP1) with disease progression [192] and [237].

“
“Monitoring sensu stricto includes the rigorous sampling of a biological, physical and/or chemical ecosystem component for a well-defined purpose and against a well-defined end-point ( McLusky and Elliott, 2004). That aim may be the detection of a trend or the

non-compliance with a threshold, standard, trigger value or baseline, thus leading to a well-defined (and agreed in advance) policy action ( De Jonge et al., 2006). In this way, aquatic and marine legislation worldwide requires adequate and rigorous monitoring at different spatial and temporal scales, such as in the Clean Water Act (CWA) and Oceans Policy (USA), the Oceans Act (Canada, Australia), the Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD) (Europe) ( Borja et al., 2008). Despite this, monitoring sensu lato selleck compound trans-isomer manufacturer has achieved many other meanings, many of which now codified in the above legislation. In a previous Editorial, we identified 10 types of monitoring ( Elliott, 2011), covering everything from wide surveillance (for which a pre-determined endpoint may not have been defined), through operational monitoring (by an industry wanting to know or demonstrate its performance), to investigative monitoring (also called diagnostic monitoring, which is better regarded as applied research possibly to find

the cause of a measured effect). Although making the concepts of monitoring more complex, each of these 10 types has been defined for a purpose – again with an intent to aid in management, to provide relevant and timely information. Edoxaban Similarly, we have indicated

the 18 characteristics of monitoring programmes and the indicators of change detected during those programmes ( Elliott, 2011). Hence, the need for a rigorous, scientifically and legally defendable approach and resulting data is clear. For example, most monitoring required by statutory agencies, especially that linked to conditions stipulated in licences/permits/consents/authorisations, have to stand up to legal scrutiny otherwise there will be legal challenges either on the developer (the industry or pollution discharger) or the regulator issuing the permissions to operate. Many Editorials and papers in Marine Pollution Bulletin have emphasised the importance of monitoring in marine waters (e.g. Tanabe, 1993, Pearce, 1998 and Wells and Sheppard, 2007). Our journal has published a total of 270 papers, having the word ‘monitoring’ in the title, since the first volume ( Holden, 1970) to the recent ones ( Purser and Thomsen, 2012). Despite the importance of monitoring, in terms of non-compliance with a threshold and the subsequent need for (expensive) policy and managerial actions, the current global economic crisis, and especially cuts in government spending, is leading many countries (and industries) trying to save money in their monitoring budgets.

minimum at a final concentration of 6000–8000 cells mL−1, in the absence (control = 0 μg mL−1

DD) or presence of decadienal (DD) at different concentrations (0.5 and 2 μg mL−1 of DD). P. minimum was used as the control diet since it does not produce aldehydes or other oxylipins. For each treatment (control, 0.5 and 2 μg mL−1 of DD), three flasks were used. Three groups of T. stylifera females (N = 5) were incubated with P. minimum in the absence of DD (control treatment). Three groups of T. stylifera females (N = 5) were incubated with P. minimum in the presence of DD at the above mentioned concentrations (experimental treatments). After 24 h of incubation at 20 °C, females were counted again in the experimental treatments and phytoplankton was fixed see more with Lugol’s solution. Samples were counted under a direct microscope in 1 mL Sedgewick–Rafter chambers. Ingestion rates (cells ind−1 h−1) were calculated following Frost’s equations ( Frost, 1972) and were then converted into μg C ind−1 h−1 considering that P. minimum carbon content was 274.19 pg C cell−1 ( Turner et al., 2001). Freshly-collected (∼2 h after collection) healthy mature T. stylifera

males (N = 12) and females (N = 12) were isolated under a Leica stereomicroscope and incubated individually in 5 mL tissue culture wells filled with 0.45-μm filtered seawater (FSW) (control) or DD at different concentrations (0.5, 1.0, 2.0, 3.0, 5.0 and 12 μg mL−1). After 24 h of incubation at 20 °C without any food, survival of males and females was assessed in the different wells. Dead copepods were counted click here in each well and the percentage of survivorship was determined for each DD concentration. In order to test the biological activity of Rutecarpine DD on T. stylifera reproduction, freshly collected (∼2 h after collection) healthy mature females (N = 10) with dark gonads ( Ianora et al., 1989) were incubated individually in 5 mL tissue culture wells filled with FSW (control) and with DD at different concentrations (0.5, 1.0 and 2.0 μg mL−1). All groups of copepods were incubated in

a temperature controlled chamber at 20 °C and 12 h:12 h light:dark cycle without any food. T. stylifera females were checked under a Leica microscope to detect egg production every half hour. After spawning, females were removed and eggs were left to hatch for 48 h; percentage egg viability was calculated as described by Ianora et al. (1995). Eggs were checked every hour to determine hatching times. After 48 h nauplii were fixed with formalin and counted under a Leica microscope. At the end of the reproduction experiments, all of the nauplii of the different replicates for each treatment (DD and controls) were pooled together for the TUNEL (terminal deoxy-nucleotidyl-transferase-mediated dUTP nick end labeling) analysis to calculate % of apoptotic nauplii with respect to total nauplii.

To study changes in relative quantity of CD184 (CXCR4) and CD62L (L-selectin) on the cell subsets, the median fluorescence intensity (MFI) of the labeled anti-CD184-antibody and anti-CD62L-antibody was analyzed. Samples for measuring hormone concentrations were kept frozen at −70 °C until assay. Cortisol in serum and adrenocorticotropic

hormone (ACTH) in plasma were measured using a commercial assay (Immulite, Siemens Healthcare Diagnostics, Deerfiled, USA). Aldosterone was measured in serum, also using GW3965 cost a commercial assay (DPC-Biermann, Bad Nauheim, Germany). Epinephrine and norepinephrine were measured in plasma by standard high-performance liquid chromatography. Sensitivity, intraassay and interassay coefficients of variation were as follows: cortisol 0.2 μg/dL, less than 10%; ACTH 9 pg/mL, less than 9.5%; aldosterone 11 pg/mL, less than 16%; epinephrine 2 pg/mL, less than 6.5%; norepinephrine 5 pg/mL, less than 6%. Sleep stages were determined off-line from polysomnographic recordings following standard criteria (Rechtschaffen and Kales, 1968). For each night, sleep onset (with

reference to lights off at 23:00 h), total sleep time (sum of time spent in sleep stages 1, 2, 3, and 4 and REM sleep), and the time as well as percentage of total sleep time spent in the different sleep stages were calculated. In addition, http://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html we determined the time between awakening of subjects around 4:00 h for the second administration of spironolactone or placebo and falling asleep again. SWS was defined by the sum of stage 3 and 4 sleep. Analyses of variance (ANOVA) for repeated measurements were calculated on T cell subpopulations (absolute counts, CXCR4 expression, CD62L expression) and hormones. Factors Arachidonate 15-lipoxygenase included were “Condition” (spironolactone versus placebo), “Early/late” (23:00–3:30 h versus 5:00–9:30 h) and ”Time” (reflecting the four time points of measurements during the early and late night, respectively). We included the factor “Early/late” since we expected the

effects of spironolactone only during the early night, when the impact of sleep on T cell migration is evident (see Section 1). Degrees of freedom were corrected according to the Greenhouse-Geisser procedure. In case of ANOVA effects, paired t tests were analyzed at single time points. A p-value <0.05 was considered significant. Data are presented as mean ± SEM. T cells were classified as CD4+ (T-helper) and CD8+ (cytotoxic) T cells, and both of these subpopulations were further divided in naïve, central memory, effector memory and effector T cells. The absolute counts of all subpopulations with the exception of effector CD4+ and effector CD8+ T cells showed a peak during the first night half (23:00–3:30 h) followed by a decline until the last blood sampling at 9:30 h (F(1,10) ⩾ 9.68, p ⩽ 0.01, for main effects of Early/late and F(3,30) ⩾ 8.27, p ⩽ 0.

marajoensis (unpublished data), cause neuromuscular blockade at very low concentrations (0.1–30 μg/ml) via a presynaptic action, as suggested by (1) quantal

content measurements in mouse diaphragm muscle and (2) the lack of effect on the muscle responses to exogenous ACh and KCl, no CK release, no significant change in the membrane resting potential and no inhibitory effect on the response to direct muscle stimulation, indicating a lack of muscle damage. Since these same characteristics were seen here with B. b. smargadina venom, we conclude that this venom also causes neuromuscular blockade by acting presynaptically, like Bothrops venoms and their toxins ( Cogo et al., 1998, Borja-Oliveira et al., 2007 and Ponce-Soto www.selleckchem.com/products/obeticholic-acid.html find more et al., 2009). Indeed, the high potency of B. b. smargadina venom (50% blockade in ∼15 min with 10 μg/ml) was similar to that of several Bothrops venoms shown to have presynaptic activity (time for 50% blockade: B. marajoensis 17 ± 1 min, B. insularis 30 ± 2 min and B. neuwiedi 42 ± 2 min)

( Rodrigues-Simioni et al., 2004 and unpublished data). The decrease in potency (increased time for 50% blockade) and the attenuation of facilitation seen when the experiments were done at 22 °C and when PLA2 activity was inhibited with BPB suggested the involvement of PLA2 activity in these responses, as also reported for Bothrops venoms ( Cogo et al., 1993 and Rodrigues-Simioni et al., 2004). However, as shown by the experiments with d-tubocurare, the neuromuscular activity of the venom in PND preparations appears to involve at least two components: one that causes prolonged facilitation (non-PLA2) and one that contributes to the initial phase

of facilitation and causes neuromuscular blockade (most likely PLA2). The incidence of bites by B. b. smargadina in humans varies considerably (3–38%) throughout its range in the Amazon basin ( Smalligan et al., 2004 and Warrell, 2004), with most bites involving the wrists, hand, arms and upper body, including the face, head and neck region because Amobarbital of the species’ arboreal habits. The clinical manifestations of envenoming by this species are similar to those of Bothrops spp., viz, local swelling, pain and bruising (but necrosis is unusual), with the main systemic responses being coagulopathy and spontaneous bleeding ( Warrell, 2004); no manifestations of neurotoxicity have been reported. This discrepancy between the results for in vitro and in vivo neurotoxicity may reflect the presence of circulating endogenous PLA2 inhibitors in human plasma. Indeed, molecules capable of inhibiting venom metalloproteinase or PLA2 activities in vitro and in vivo have been isolated from snake and mammalian (opossum) plasma ( Lizano et al., 2003), and human plasma contains a PLA2 inhibitor ( Miwa et al., 1984 and Miwa et al., 1985).

The spatial dynamics of fishery resources (notably the key sea cucumber and spiny lobster stocks) and of the fishing fleet must be measured and modeled to assess the applicability of spatially-explicit management measures (TURFs, seasonal closures, spatial gear restrictions,

etc.) in order to reduce overexploitation risks. Consider, for Inhibitor Library manufacturer example, the case of broadcast spawners, such as sea cucumbers, which – as for many sedentary species – require high density concentrations in order to reproduce successfully. Such high-density patches are the first to be targeted by fishers in a fishery regulated by catch or effort limits [37], making management measures such as total allowable catch (TAC) inappropriate in the fisheries for these species. In this case, a spatially explicit management tool, such as seasonal closures, could be more effective than a TAC (e.g., to protect sea cucumber juveniles). On the other hand, caution is needed with spatial measures such as no-take zones since changes in the distribution of fishing effort could lead to overfishing

of the stocks located outside the zone [37] and [52]—it is thus necessary to evaluate the impact of zoning on fleet distribution. Current monitoring Selleck Sunitinib programs must be evaluated, adapted, and coordinated with the goal of producing needed spatial planning information, integrating the collection of socioeconomic data on a regular and strategic basis. According to Day [11], the establishment of a robust monitoring system to evaluate the effectiveness of marine spatial management plans requires a major institutional reorientation at the policy

level. In the case of Galapagos, it will require a major adaptation of the GMRMP, including as a priority the allocation of suitably long-term governmental Buspirone HCl funding to ensure the continuity and efficiency of the monitoring programs. Also important are efforts to better utilize existing data (biophysical, socioeconomic and fishery data) in order to extract the maximum value from them [44]. Furthermore, the above-noted monitoring capability of VMS together with the recent implementation of an Automatic Identification System (AIS) for the entire local fishing fleet, provides an unique opportunity to better understand the spatial behaviour of fishers, and thereby to predict how this behaviour interacts with spatial population processes to determine the character of exploited meta-populations; and to understand the implications of policy options ranging from no-take zones to TURFs [43]. Such an evaluation of the GMR will facilitate adaptation of the marine zoning scheme, taking into consideration the scientific information available, the local fishery knowledge and the lessons learned as outlined above.

The homogenates were centrifuged for 30 min at 20,000 × g at 4 °C. The supernatants were recovered and the pellets (except those of midgut contents) were resuspended in double-distilled water. The pellets are regarded as cell membrane fractions. The samples were stored at −20 °C until use. No enzyme inactivation was detected during storage. Midgut section contents (V1, V2 + V3, and V4), isolated as described above, were dispersed

in 5 μl of the dissecting saline and added to 5 μl of a 5-fold dilution of a universal pH indicator (E. Merck, Darmstadt, pH 4–10). The resulting colored solutions were compared with appropriate standards. Protein was determined based on the method described by Bradford (1976), using ovalbumin as a standard. General proteolytic activity was determined with two different substrates: 0.5% (w/v) fluorescein isothiocyanate-labeled (FITC) casein

(casein-FITC) (fluorescent substrate, useful at TSA HDAC pH values above 5) or 0.5% hemoglobin-FITC (fluorescent substrate, useful at pH values below 4.5). The preparation of the substrates and the assays was based on the method described by Twining (1994) in 50 mM sodium citrate-phosphate buffer at pH 5.5 with casein-FITC or in the same buffer this website at pH 3.5 with hemoglobin-FITC as substrate. Unless otherwise specified, other proteinase assays were carried out in 50 mM sodium citrate-phosphate buffer, pH 5.5, with the following fluorescent substrates: 10 μM carbobenzoxy-Phe-Arg-7-amino-4-methyl Oxaprozin coumarin (Z-FR-MCA) (substrate for trypsin); 10 μM succinyl-Ala-Ala-Phe-MCA (S-AAF-MCA) (selective substrate for chymotrypsin); and 1 μM ɛ-amino-caproyl-leucyl-(S-benzyl) cysteinyl-MCA (selective substrate for cysteine proteinase). With these substrates, proteinase activity was measured by methylcoumarin fluorescence (excitation 380 nm and emission 460 nm). Inhibitors/activators were used

at the following final concentrations: trans-epoxysuccinyl-l-leucyl-amido (4-guanidino butane) (E-64), 10 μM; benzamidine, 0.25 mM; EDTA, 5 mM; pepstatin A, 1 μM; chymostatin, 25 μM; EDTA/DTT, 3/1.5 mM; and soybean trypsin inhibitor (SBTI), 17 μM. These substances were pre-incubated with the supernadant of whole midgut homogenates at room temperature for 15 min before adding the substrate. Unless otherwise specified, aminopeptidase, amylase and maltase were determined as follows: aminopeptidase was assayed in 50 mM Tris–HCl buffer (pH 7.0) using 1 mM l-leucyl-p-nitroanilide (LpNA), based on the method described by Erlanger et al. (1961); amylase was measured by determining the appearance of reducing groups ( Noelting and Bernfeld, 1948) in 50 mM sodium citrate-phosphate buffer at pH 6.0 with 0.5% (w/v) starch as substrate in the presence of 10 mM NaCl; and maltase was assayed based on the method described by Dahlqvist (1968), using 7 mM maltose in 50 mM sodium citrate-phosphate buffer at pH 6.0.