Decrease In Protease Activity Research Articles

Impaired heterologous protein-protein interaction is an essential cause for non-viability of WNV/DENV recombinants

Flavivirus RNA replication starts at 3′-end, where it folds into a highly conserved stem-loop structure. We attempted to identify the viral non-structural proteins (NSPs) that might specifically interact with the 3′-stemloop (3′SL) through a genetic approach. WNV/DENV2 chimeric recombinants that contain Dengue2 (DENV2) gene(s) in West Nile virus (WNV) backbone were tested for replication competence. Three of seven recombinant viruses, containing the DENV2 NS1, NS2A, or NS4B gene and terminated with a mutated 3′SL (MutC 3′SL), were viable. Of these three, only those bearing the DENV2 NS1 and NS2A substitutions remained infectious when the MutC 3′SL was replaced by the wildtype WNV 3′SL. However, none of the seven chimeric recombinants bearing the DENV2 3′SL were viable. We then investigated the causes for failed replication of WNV/DENV2 chimeric recombinants. Proteolytic cleavage of NS polyproteins was defective by heterologous protease NS2B/3, but was efficient by homologous DENV2 NS2B/3 protease. Whereas, the heterologous polyproteins that contained DENV2 homologous protease were found to produce abnormal vesicles. WNV/DENV2 recombinants expressing the DENV2 homologous protease did not produce infectious virus either. We examined NS protein-protein interaction (PPI) and found that heterologous PPI (hPPI) between WNV and DENV2 NSPs were impaired to various degrees. Insufficient PPIs occurred mainly between heterologous NS2B and NS3; NS2B and NS4A; NS3 and NS5, correlating to those non-viability of substitution mutants. Our results indicate that impaired PPI may decrease protease activity and affect vesicle formation, and is the essential cause for non-viability of the WNV/DENV2 recombinants.

Expression of cystatin C in human esophageal cancer.

Cystatin C is a member of the cysteine protease inhibitors and its function is to decrease protease activity. A recent study showed that it was aberrantly expressed in many malignant tumors in association with tumor invasion and metastasis. We attempted to detect its expression in esophageal cancer tissues and adjacent reparative normal tissues. Samples of cancers and non-cancerous esophageal tissues were obtained as matched pairs from 30 surgery patients with esophageal cancer and paraffin embedded. The expression of cystatin C in tissues was investigated by immunohistochemistry. Fisher's exact test was used to analyze the relationship between esophageal cancer tissues and adjacent normal tissues. Furthermore, mRNA was extracted, and reverse transcriptase polymerase chain reaction was performed. The intensity of cystatin C immunostaining in tumor tissues was increased compared to that of adjacent normal tissues. mRNA expression of the cystatin C gene was greater in esophageal cancer than in normal tissues (P <0.05). Our results indicate that cystatin C may play an important role in the pathogenesis and metastasis of esophageal cancer.

PH-induced conformational change of natural cyclic lipopeptide surfactin and the effect on protease activity

The cyclic lipopeptide surfactin (SF) is one of the promising environmental friendly biosurfactants abundantly produced by microorganisms such as Bacillus subtilis. SF shows excellent surface properties at various pH, together with lower toxicity and higher biodegradability than commonly used petroleum-based surfactants. However, the effect of the dissociation degree of SF on self-assembly is still incompletely understood, even though two acidic amino acid residues (Asp and Glu) are known to influence eventual surface and biological functions. Here, we report changes in the secondary structure of SF induced by increased pH, and the effect on protease activity. We found that the β-sheet and β-turn formation of SF are significantly enhanced through increased dissociation of Asp and Glu as revealed by a titration experiment of SF solution to estimate apparent pK1 and pK2 values together with circular dichroism spectroscopy. We also studied the activity of the common detergent enzyme subtilisin in SF solution at above its pK2 (pH 7.6) to understand the role of the dissociation degree in the interaction with the protein. The mixing of SF having a unique cyclic topological feature with subtilisin suppressed the decrease in protease activity observed in the presence of synthetic surfactants such as sodium dodecyl sulfate and polyoxyethylene alkyl ether. Thus, SF has great potential for use in laundry detergent formulations, to improve the stability and reliability of detergent enzymes.

Abstract TMP92: Characteristic Brain MRI Features of Manifesting Heterozygotes With Cerebral Autosomal Recessive Arteriopathy With Subcortical Infarcts and Leukoencephalopathy

Introduction: Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a cerebral small-vessel disease (CSVD). Mutations in the high-temperature requirement serine peptidase A1 gene ( HTRA1 ) cause CARASIL via a decrease in protease activity of HTRA1. Although most of the heterozygotes with the HTRA1 mutation are healthy, manifesting heterozygotes have been reported. We have elucidated that the mutant HTRA1s that develops CSVD in a heterozygote state have a distinct molecular mechanism, resulting in the dominant negative effect. These individuals showed mild phenocopy of CARASIL. However, it is not clear whether brain MRI findings in manifesting heterozygotes are different from those of CARASIL. In this study, we aimed to clarify the characteristic brain MRI features in manifesting heterozygotes by comparing them to those in CARASIL. Methods: We have evaluated 19 MRIs in eight manifesting heterozygotes and 21 MRIs in seven CARASIL patients and scored the MRIs by using a semi-quantitative scale for CARASIL, which scored white matter lesions (WMLs) (signal score) and atrophy (atrophy score) (Nozaki et al. Neurology 2015). Statistical analysis was conducted using software R 3.2.2. We obtained written informed consent from all individuals. Results: Signal score in manifesting heterozygotes was significantly lower than that in CARASIL (Mean ± SD; 14.6 ± 1.9 vs. 23.1 ± 5.0, p &lt; 0.0001), however, there was no difference in atrophy score between the two groups (Mean ± SD; 5.5 ± 2.2 vs. 7.5 ± 5.5, p = 0.20). Atrophy score showed positive correlation with the disease duration in both groups (r 2 = 0.48, p = 0.0014 vs r 2 = 0.41, p = 0.0041), however signal score showed no correlation with the disease duration. Conclusion: WMLs is milder in manifesting heterozygote as compared with CARASIL. In contrast, the brain atrophy is not influenced by the HTRA1 mutation status but positively correlated with the disease duration. The rate of carriers for pathogenic HTRA1 mutations are higher than expected. These characteristic findings of brain MRIs might be useful to pick up the candidate for the genetic screening for HTRA1 .

The Antihypertension Effect of Fermented Skipjack Tuna (Katsuwonus pelamis L.)/Bakasang’s Peptide Extract Based on Cardiac’s Histopathology and Protease Activity on Hypertensive Rats Induced by Deoxycorticosterone Acetate (DOCA) -Salt

Hypertension is an abnormality of cardiovascular condition, marked by high-blood pressure over the normal condition. One of the factors that causes hypertension is altered equilibrium of Renin-Angiotensin-Aldosteron system (RAAS). This condition, which commonly by genetic could causes excess production of Angiotensin II, which known as vasoconstrictor, carried out by Angiotensin Converting Enzyme (ACE). Angiotensin II could increases the body’s blood pressure, and stimulates the production of free radicals, causing hypertension and oxidative stress. One of the common medication for hypertensive patient are using ACE inhibitor pills. Consumption of commercial pills, had a negative effect on the patient, such as skin iritation, cough, allergic reaction, etc. This study was conducted to explore the potential peptide of bakasang extract isolated from fermented skipjack tuna (Katsuwonus pelamis L.) for antihypertensive therapy, based on the activity of protease and histopathology of cardiac tissue on hypertensive rats (Rattus norvegicus) induced by DOCA-salt. The rats were divided into five groups: (1) negative control group, (2) hypertensive control group, (3) hypertension with captopril therapy dose of 5 mg/kg of body weight (BW), (4) and (5) hypertension with bakasang’s peptide extract therapy dose of 200 mg/kgBW, and 300 mg/kgBW, respectively. The results showed that bakasang’s peptide extract dose of 200 mg/kgBW significantly (p less than 0.05) decrease protease activity and improve histopathology of cardiac tissue on hypertensive rats. Protease activity post-therapy decreased to be 39.27 % from positive hypertension group. It could be concluded that the peptide of bakasang’s peptide extract has antihypertension effect (ACE-inhibitors), which was able to improve histopathology and decrease protease activity of the cardiac tissue on hypertensive rats induced by DOCA-salt.

Changes in non-specific proteases and their inhibitors in the course of acute purulent rhinosinusitis treatment with mucoactive drugs

Objective of the study is to determine the influence of mucoactive drugs of various groups on the activity of non-specific proteases and their inhibitors in the blood serum and sinonasal discharge during treatment of patients with acute purulent rhinosinusitis. Materials and methods. 48 patients with acute purulent rhinosinusitis were treated; they were randomly assigned to four groups: N-acetylcysteine was used in Group 1 (n = 11), S-carboxymethylcysteine – in Group 2 (n = 14), phytoneering herbal drug (BNO-101) Sinupret – in Group 3 (n = 13). The control group consisted of patients (n = 10) who did not receive mucoactive agents during the treatment. Normal values were determined in 12 healthy volunteers. The material for the study included blood serum and sinonasal discharge of the patients. We studied elastase-like activity, trypsin-like activity, antitryptic activity and acid-stable inhibitors. Results. Conducted treatment course resulted in improved clinical symptoms and decreased proteolytic activity in the blood serum and sinonasal discharge of the patients with acute purulent rhinosinusitis as compared to the patients’ parameters before treatment. Against this background, the state of proteinase inhibitors did not significantly change. This is primarily caused by the fact that antitryptic activity in acute purulent rhinosinusitis remained practically unchanged as compared to the parameter at the beginning of treatment. However, there is a tendency towards an increase in the inhibitory potential during treatment with mucoactive drugs. The highest level of this increase was observed when patients were taking BNO-101. On Day 10 of the study it resulted in the significant increase in antitryptic activity(ATA) as compared to the group prior to the treatment. The level of acid-stable inhibitors (ASI) in patients with purulent rhinosinusitis significantly decreased in the settings of acute purulent inflammation, and the activity of ASI increased during the treatment. Conclusion. The study of non-specific proteases and antiprotease potential of sinonasal discharge demonstrates that the decrease in protease activity and normalization of their inhibitors level depends on the mucoactive agent chosen. The most effective treatment option reduces the aggressive effect of non-specific proteases on mucosa and connective tissue structures. The mucoactive drugs’ effect of improved drainage increases antiprotease potential by restoring the structure and functional activity of the mucosa. Restoration of mucosa increases synthesis and secretion of acid-stable protease inhibitors which are the key component of local antiprotease protection.

Quorum sensing-disrupting coumarin suppressing virulence phenotypes in Vibrio splendidus.

In the present study, the effects of an environmental friendly natural reagent coumarin, on the growth and potential virulence factors, as well as its ability to interfere the infection of Vibrio splendidus (Vs), were determined. Coumarin showed no effects on the maximal growth of Vs, and biofilm formation of Vs, while it significantly decreased protease activity and hemolytic activity by 43 and 80%, respectively. Correspondingly, coumarin exhibited an obviously protective effect, with a relative percent survival of 60% upon Apostichopus japonicus from infection by Vs. To preliminarily investigate the mechanism underlining the inhibitory effects, regulation of genes Vsm and Vsh respectively related to protease activity and hemolytic activity by supernatant and supernatant extract containing acyl-homoserine lactones (AHLs) and coumarin was determined. Cell-free supernatant from higher density and its ethyl acetate extract containing AHL signal molecules could respectively upregulate the mRNA level of Vsm by 17.4- and 2.3-fold and Vsh by 7.2- and 5.0-fold, when Vs was at lower cell density. However, coumarin could reduce the stimulatory effects of both the supernatant and its ethyl acetate extract. Combining all the results in our study, it was suggested that coumarin could be considered as an alternative to be used for controlling infection of Vs, downregulating the expression of potential virulence factors through interfering the AHL-mediated pathways.

Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells

Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos

Brown bears (Ursus arctos) hibernate for 5–7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

Transgenic Expression of a Viral Cystatin Gene CpBV-CST1 in Tobacco Confers Insect Resistance.

A viral gene, CpBV-CST1, was identified from a polydnavirus Cotesia plutellae bracovirus (CpBV). Its protein product was significantly toxic to lepidopteran insects. This study generated a transgenic tobacco plant expressing CpBV-CST1 Expression of transgene CpBV-CST1 was confirmed in T1 generation (second generation after transgenesis) in both mRNA and protein levels. Young larvae of Spodoptera exigua (Hübner) suffered high mortalities after feeding on transgenic tobacco. All 10 T1 transgenic tobacco plants had no significant variation in speed-to-kill. In order to further explore insect resistance of these transgenic tobaccos, bioassays were performed by assessing antixenosis and antibiosis. S. exigua larvae significantly avoided T1 plants in a choice test. Larvae fed with T1 plant exhibited significant decrease in protease activity in the midgut due to consuming CpBV-CST1 protein produced by the transgenic plant. Furthermore, the transgenic tobacco exhibited similar insect resistance to other tobacco-infesting insects, including a leaf-feeding insect, Helicoverpa assulta, and a sap-feeding insect, Myzus persicae These results demonstrate that a viral cystatin gene can be used to develop insect-resistant transgenic plant, suggesting a prospective possibility of expanding the current transgenic approach to high-valued crops.

Oxidation and Tyrosine Nitration Induce Structural Changes and Inhibits Plasmodium falciparum Falcipain-2 Activity In Vitro.

Falcipain-2 (FP2) is an important hemoglobinase from the malaria parasite Plasmodium falciparum and a suitable target for the development of an antimalarial chemotherapy. Many reports have indicated that radical nitrogen species (RNS) including nitric oxide (NO) are inhibitors of P. falciparum growth and promoters of recovery from malaria symptoms. In this scenario, FP2 emerges as a potential target of RNS, since its inhibition partially hinders the parasite growth. We report that in vitro FP2 did not undergo S-nitrosylation when exposed to the NO-donor GSNO. However, it was modified by a combined mechanism of methionine oxidation and tyrosine nitration in response to SIN-1, and NaNO2- H2O2 treatment. The treatments with the nitrating agents caused a pronounced decrease in protease activity most likely induced by a disruption on the secondary and tertiary structure of FP2. Our data also demonstrate that at least four tyrosine residues were nitrated and found on the surface of the enzyme, partially or completely exposed to the solvent. Although performed in vitro, these results suggest that falcipain-2 may be a target of RNS activity and its inhibition could explain the hindering of the parasite growth when exposed to these radicals. The understanding of the molecular mechanisms involving free radicals and its inhibition activity towards FP2 may be effective in the development of antimalarial therapies.

Pod removal responsive change in phytohormones and its impact on protein degradation and amino acid transport in source leaves of Brassica napus

To characterize the hormonal regulation of nitrogen remobilization from source to pod filling in Brassica napus, the hormonal level, proteolytic process, and amino acid transport were assessed in mature leaves of pod-removed or control at the early pod-filling stage. Pod (sink) removal decreased salicylic acid (SA), and significantly increased jasmonic acid (JA). The SA/JA ratio decreased with pod removal, accompanied by low degradation of foliar proteins and Rubisco content. A significant decrease in protease activity was observed in pod-removed leaves, confirmed by in-gel staining of protease. Pod removal reduced the expression of four amino acid transporter genes (BnAAP1, BnAAP2, BnAAP4, and BnAAP6) in mature leaves and reduced amino acid loading into phloem. These results indicated that a decrease in SA resulting from pod removal down-regulated nitrogen remobilization accompanied by a decrease in proteolytic activity and amino acid transport in mature leaves at the pod-filling stage.

The phenotypic evolution of Pseudomonas aeruginosa populations changes in the presence of subinhibitory concentrations of ciprofloxacin.

Ciprofloxacin is a widely used antibiotic, in the class of quinolones, for treatment of Pseudomonas aeruginosa infections. The immediate response of P. aeruginosa to subinhibitory concentrations of ciprofloxacin has been investigated previously. However, the long-term phenotypic adaptation, which identifies the fitted phenotypes that have been selected during evolution with subinhibitory concentrations of ciprofloxacin, has not been studied. We chose an experimental evolution approach to investigate how exposure to subinhibitory concentrations of ciprofloxacin changes the evolution of P. aeruginosa populations compared to unexposed populations. Three replicate populations of P. aeruginosa PAO1 and its hypermutable mutant ΔmutS were cultured aerobically for approximately 940 generations by daily passages in LB medium with and without subinhibitory concentration of ciprofloxacin and aliquots of the bacterial populations were regularly sampled and kept at - 80 °C for further investigations. We investigate here phenotypic changes between the ancestor (50 colonies) and evolved populations (120 colonies/strain). Decreased protease activity and swimming motility, higher levels of quorum-sensing signal molecules and occurrence of mutator subpopulations were observed in the ciprofloxacin-exposed populations compared to the ancestor and control populations. Transcriptomic analysis showed downregulation of the type III secretion system in evolved populations compared to the ancestor population and upregulation of denitrification genes in ciprofloxacin-evolved populations. In conclusion, the presence of antibiotics at subinhibitory concentration in the environment affects bacterial evolution and further studies are needed to obtain insight into the dynamics of the phenotypes and the mechanisms involved.

Rising atmospheric CO2: Potential impacts on chickpea seed quality

Experiments on chickpea (Cicer arietinum L.) were performed in open-top chambers during 2010–11 and 2011–12 to assess effects of atmospheric CO2 enrichment on the quality of seeds. Although no physical modification was observed, an increase in seed water uptake was recorded in plants grown under enriched atmospheric CO2 condition. Germination of seeds reduced by 45–47%, while seed leachate conductivity increased by 10–17%. Seedling vigor decreased, although root and shoot lengths and seedling biomass showed negligible changes. Similarly, atmospheric CO2 enrichment reduced field emergence of seedlings with no change in root characteristics of the emerged seedlings. A decrease in protease activity supports the reduced seed viability, although no change in grain phosphatase and α-amylase activities were recorded. Increase in carbon content in germinating seed-cotyledon along with decrease in N in cotyledon resulted in large increase in C:N ratio for the plants grown under enriched CO2 condition. The starch content increased with no change in soluble sugar in germinating seed-cotyledons. This indicates more carbonaceous seeds from plants grown under enriched CO2 environment. Results suggest that rising atmospheric CO2 might have adverse impact on viability and germination of chickpea seeds, and cause nutritional imbalance through increase in C with dilution of N contents in germinating seed-cotyledons.

0270: TREM-1 is a therapeutic target during myocardial infarction

The innate immune system has recently been shown to be important in cardiac response to MI. TREM-1 is an immune-receptor expressed by neutrophils, macrophages, and mature monocytes that acts as an amplifier of the innate immune response triggered by Toll-Like Receptors (TLRs) engagement. We here aim at investigating the mechanisms by which LR12 confers protection. We used adult male mice TREM-1 -/- , Rag1 -/- (lacking B and T cells) and mice depleted in neutrophils with anti-Ly6G 1A8. Animals were submitted to a permanent MI and then randomized to receive LR12 (a synthetic TREM-1 inhibitory peptide), LR12 scramble or anti- TREM-1 mAb (as a TREM-1 agonist) ip beginning 1 hour after MI, then daily for 5 days. In different types of mice, we first observed that TREM-1 was expressed (except in TREM-1 -/- ) in myocardial tissue, especially in infarcted areas with an expression peaking at 24h after MI. We next observed that in TREM-1 -/- and LR12 treated animals infarct size was reduced. Recruitment of inflammatory cells (neutrophils, Ly6C hi monocytes, B cells) was also reduced, while ‘anti-inflammatory’ Ly6C low monocytes number was increased. In LR12 treated and TREM-1 -/- mice we observed a reduction of leucocytes activation. Using both qRT-PCR and zymography we found that deletion or modulation of TREM-1 was associated with a decreased protease activity in infarcted areas (MMP9/TIMP1). Finally, while activation of TREM-1 through anti- TREM-1 mAb administration decreased survival, LR12 and deletion of TREM-1 conferred protection. The phenomena obtained in TREM-1 -/- and LR12 treated was also observed in Rag1-/-and mice depleted in neutrophils. TREM-1 plays an important role in mediating inflammatory cells recruitment and activation following myocardial infarction. Its therapeutic modulation achieved through LR12 administration confers protection in mice.

Niclosamide blocks autophagy via lysosomal dysfunction (663.18)

Niclosamide is a salicylanilide derivative used as antihelminthic agent. Recently, Nic has been screened out as an autophagy modulator by us and other groups and was identified to be a structurally distinct mTORC1 signaling inhibitor. In this work, we measured the long‐lived protein degradation activity and found Niclosamide could inhibit the autophagic process. To elucidate the inhibition of Niclosamide on autophagy, we further tested the lysosomal activity using DQ‐BSA assay and measured protease activity such as Cathepsin B and Cathepsin D with fluorescent substrates. The lower degradation of DQ‐BSA and decreased protease activity after Niclosamide treatments indicated the impaired lysosome function. In addition, both LC‐II and P62 proteins accumulated significantly when treated by Niclosamide suggesting the late stage of autophagy was blocked. The increased pH of lysosome and release of Cathepsins from lysosome to cytosol demonstrated that Niclosamide might affect lysosome resulted from lysosomal membrane permeabilization. Collectively, we firstly disclosed Niclosamide not only inhibited mTORC1 activity but also, as other well‐known lysosome inhibitors, strongly blocked autophagy via lysosomal dysfunction.Grant Funding Source: Supported by Guandong Provincial Natural Science Foundation, China (S2013010015876)

Hypothetical cathepsin-like protein from Nematostella vectensis and its silicatein-like cathepsin mutant for biosilica production

Silicatein has high sequence identity and similarity with that of cathepsin L. In silicatein, serine replaces the active-site cysteine that is found in cathepsin L. Here, we obtained hypothetical cathepsin-like protein (CAT) from Nematostella vectensis which is 55% identical and 75% similar to mature silicatein alpha (SIL) of Suberites domuncula. When this protein was expressed in Escherichia coli, it displayed protease activity with both N-carbobenzoxy-l-phenylalanyl-l-arginine-4-methylcoumaryl-7-amide (Z-FR-AMC) and gelatin substrates, as well as silica-condensing activity using the tetraethoxy silane (TEOS) substrate. To increase its silica-forming activity and stability, some residues including the active site cysteine, were mutated into conserved silicatein residues, resulting in a mutant with 65% identity and 79% similarity to SIL. The mutant silicatein-like cathepsin (SLC) had increased expression levels in E. coli, and silica-forming activity comparable to that of SIL. In addition, SLC exhibited decreased protease activity as compared to that of CAT. Both CAT and SLC produced silica particles of sizes smaller than 50nm, which increased to 200–300nm in the presence of a structure-directing agent, such as Triton X-100. In conclusion, CAT was evolved to function as a biosilica-forming protein, and SLC was engineered by mutating CAT residues into conserved SIL residues to produce various silica-based materials.

Protective effects of TREM-1 modulation during experimental myocardial infarction

Purpose: Myocardial healing after infarction is hampered by reperfusion injury and ventricular remodeling, partly mediated by detrimental inflammatory responses. We hypothesized that modulation of the Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), an amplifier of the innate inflammatory response, by the use of a small synthetic peptide, LR12, could improve myocardial injury in a rodent model of myocardial infarction (MI). Methods: For hemodynamic assessment, we used Wistar rats which underwent either permanent occlusion of the left coronary artery or transient ischemia for one hour followed by reperfusion. They were randomly assigned to receive LR12 or vehicle for 5 days. Cardiac function and dimensions were assessed at baseline through positron emission tomography (PET) imaging and 6 weeks after infarction by PET and conductance catheter. For biological investigations, we used male Balb/c mouse with permanent MI and infusion of LR12 or vehicle from 1 hour after MI to day 5. TREM-1, protease activity and others myocardial inflammatory mediators tissue expression were quantified by western-blot, Elisa, and qRT-PCR. Leukocytes populations were studied in heart and blood samples. Results: Following permanent ischemia, LR12 reduced ventricular remodeling, as assessed by a lesser increase of end-diastolic volume measured by PET, and improved the systolic function evaluated by conductance catheter derived load-independent parameters (ESPVR: 0.98±0.43 vs 1.45±0.49 mmHg.μL-1, p=0.04; PRSW: 55±23 vs 80±34 mmHg.μL-1, p=0.03). During transient ischemia, LR12 infusion just before and for 5 days after reperfusion also improved myocardial contractility (PRSW: 64±25 vs 96±26, p=0.02; ESPVR 1.00±0.30 vs 1.61±0.69, p=0.05). TREM-1 was expressed in myocardial tissue, especially in infarcted areas, as early as 6 hours after MI, peaking at 24h. LR12 administration modulated myocardial inflammatory response with a reduction of p38 and ERK1/2 phosphorylation, iNOS and Cox2 expression, while Akt, GSK3β and Socs3 were up-regulated. LR12 modulated the expression of many chemokines (IL-6, -13, -17, TNF-α, ICAM-1), and decreased protease activity in myocardial infarcted areas (MMP9/TIMP1). LR12 profoundly modified leukocytes populations, with an increased number of circulating and myocardial non classical monocytes (Gr1-low) and LT4 at 96h post MI. Conclusion: LR12, by inhibiting TREM-1, improves ventricular remodeling and myocardial contractility in various experimental models of myocardial infarction. These effects are mediated by a profound switch in leukocytes recruitment to the healing myocardium.

Proteolytic activities in Phaseolus vulgaris cotyledons under copper stress

The changes in the protease activities of bean cotyledons were investigated in response to copper stress. Assays using synthetic substrates and specific protease inhibitors followed by activity measurements and electrophoresis analysis allowed to study the classes of enzymes involved in the storage protein mobilization during the germination of bean (Phaseolus vulgaris L) seeds, and then identify which ones were affected in the presence of 200μM CuCl2 in the imbibition medium. Copper treatment affected embryo growth and total protease activity. The results of SDS-gelatin-PAGE show that Cu excess led to a decrease in protease activity of 45 to 66kDa. Moreover, cysteine-, aspartic- and metallo-protease activities were markedly lowered under copper stress, while serine-protease one was enhanced as well as its activity dependent abundance in comparison with control. However, the relative distribution of major cysteine protease in H2O-germinated seeds was significantly diminished after Cu exposure. Thus, copper excess can disturb the nitrogen freeing from reserve tissues at enzymatic level; differential responses of protease classes are discussed, notably, cysteine protease in the way of storage protein mobilization and serine protease in protective mechanism one.

Impact of QseBC system in c-di-GMP-dependent quorum sensing regulatory network in a clinical isolate SSU of Aeromonas hydrophila

Our earlier studies showed that AhyRI- (AI-1) and LuxS-based (AI-2) quorum sensing (QS) systems were positive and negative regulators of virulence, respectively, in a diarrheal isolate SSU of Aeromonas hydrophila. Recently, we demonstrated that deletion of the QseBC two-component signal transduction system (AI-3 QS in enterohemorrhagic Escherichia coli) also led to an attenuation of A. hydrophila in a septicemic mouse model of infection, and that interplay exists between AI-1, AI-2, and the second-messenger cyclic-di-guanosine monophosphate (c-di-GMP) in modulating bacterial virulence. To further explore a network connection between all of the three QS systems in A. hydrophila SSU and their cross talk with c-di-GMP, we overproduced a protein with a GGDEF domain, which increases c-di-GMP levels in bacteria, and studied phenotypes and transcriptional profiling of genes involved in biofilm formation and motility of the wild-type (WT) A. hydrophila and its ΔqseB mutant. Over-expression of the GGDEF domain-encoding gene (aha0701h) resulted in a significantly reduced motility of the WT A. hydrophila similar to that of the ΔqseB mutant. While enhanced protease production was noted in WT A. hydrophila that had increased c-di-GMP, no enzymatic activity was detected in the ΔqseB mutant overexpressing the aha0701h gene. Likewise, denser biofilm formation was noted for WT bacteria when c-di-GMP was overproduced compared to its respective control; however, overproduction of c-di-GMP in the ΔqseB mutant led to reduced biofilm formation, a finding similar to that noted for the parental A. hydrophila strain. These effects on bacterial motility and biofilm formation in the ΔqseB mutant or the mutant with increased c-di-GMP were correlated with altered levels of fleN and vpsT genes. While we noted transcript levels of qseB and qseC genes to be increased in the ahyRI mutant, down-regulation of the ahyR and ahyI genes was observed in the ΔqseB mutant, which correlated with decreased protease activity. Finally, an enhanced virulence of WT A. hydrophila with increased c-di-GMP was noted in a mouse model when compared to findings in the parental strain with vector alone. Overall, we conclude that cross talk between AI-1 and QseBC systems exists in A. hydrophila SSU, and c-di-GMP modulation on QseBC system is dependent on the expression of the AI-1 system.