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  1. Fulltext Capillary-seeding crystallization and preliminary crystallographic analysis of a solvent-tolerant elastase from Pseudomonas aeruginosa strain K
    Ali MS, Said ZS, Rahman RN, Chor AL, Basri M, Salleh AB
    Int J Mol Sci, 2013 Aug 28;14(9):17608-17.
    PMID: 23989606 DOI: 10.3390/ijms140917608
    Seeding is a versatile method for optimizing crystal growth. Coupling this technique with capillary counter diffusion crystallization enhances the size and diffraction quality of the crystals. In this article, crystals for organic solvent-tolerant recombinant elastase strain K were successfully produced through microseeding with capillary counter-diffusion crystallization. This technique improved the nucleation success rate with a low protein concentration (3.00 mg/mL). The crystal was grown in 1 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic dihydrate pH 5.6. The optimized crystal size was 1 × 0.1 × 0.05 mm³. Elastase strain K successfully diffracted up to 1.39 Å at SPring-8, Japan, using synchrotron radiation for preliminary data diffraction analysis. The space group was determined to be monoclinic space group P12(1)1 with unit cell parameters of a = 38.99 Ǻ, b = 90.173 Å and c = 40.60 Å.
    Matched MeSH terms: Pancreatic Elastase/chemistry*
  2. Biochemical and gene expression studies reveal the potential of Aspergillus oryzae-fermented broken rice and brewers' rice water extracts as anti-photoageing agents
    Jamaluddin A, Mohd Abd Rahman SM, Abd Manan M, Abd Razak DL, Abd Rashid NY, Abd Ghani A, et al.
    Cell Mol Biol (Noisy-le-grand), 2023 Nov 15;69(11):9-16.
    PMID: 38015547 DOI: 10.14715/cmb/2023.69.11.2
    In this study, UVA- and UVB-irradiated human fibroblasts were used to investigate the anti-photoaging efficacy of two aqueous extracts from Aspergillus oryzae-fermented broken rice (FBR) and brewers' rice (FBrR). As UVA and UVB can damage the dermal and epidermal layers, respectively, two UV radiation approaches were utilised: i) direct UVA irradiation on fibroblasts, and ii) UVB-irradiated keratinocytes indirectly co-cultured with fibroblasts to observe their epithelial-mesenchymal interaction during UVB-induced photoaging. The anti-photoaging properties were tested utilising biochemical tests and quantitative polymerase chain reaction (qPCR). The treatment of UV-irradiated human fibroblasts with FBR and FBrR dramatically downregulates MMP-1 and SFE gene expression. Nonetheless, MMP-1 secretion was inhibited by FBR and FBrR, with more substantial decreases in UVB-treated co-cultures, ranging from 0.76- to 1.89-fold relative to the untreated control. In UVA-treated fibroblasts, however, the elastase-inhibiting activity of FBR and FBrR is up to 1.63-fold and 2.13-fold more potent, respectively. In addition, post-UV irradiation treatment with FBR and FBrR was able to repair and enhance collagen formation in UVA-irradiated fibroblasts. Both FBR and FBrR were able to upregulate elastin gene expression in fibroblasts under both culture conditions, especially at 50 µg/mL. The pro-inflammatory cytokines TNF-, IL-1ß, and IL-6 were likewise lowered by FBR and FBrR, which may have contributed to the anti-photoaging effect of the UVB-treated co-culture. These results reveal that FBR and FBrR inhibit photoaging in human fibroblasts under both UV induction conditions. In conclusion, FBR and FBrR may be attractive bio-ingredients for usage in the cosmetic sector as cosmeceuticals.
    Matched MeSH terms: Pancreatic Elastase
  3. Synthesis and characterization of new 4H-chromene-3-carboxylates ensuring potent elastase inhibition activity along with their molecular docking and chemoinformatics properties
    Dige NC, Mahajan PG, Raza H, Hassan M, Vanjare BD, Hong H, et al.
    Bioorg Chem, 2020 07;100:103906.
    PMID: 32422387 DOI: 10.1016/j.bioorg.2020.103906
    A new series of 4H-chromene-3-carboxylate derivatives were synthesized using multicomponent reaction of salicylaldehyde, ethyl acetoacetate and dimedone in ethanol with K3PO4 as a catalyst at 80 °C. The structures of all newly synthesized compounds were confirmed by spectral techniques viz. IR, 1H NMR, 13C NMR, and LCMS analysis. The newly synthesized compounds 4a to 4j were screened against elastase enzyme. Interestingly, all these compounds found to be potent elastase inhibitors with much lower IC50 value. The compound 4b was found to be most potent elastase inhibitor (IC50 = 0.41 ± 0.01 µM) amongst the synthesized series against standard Oleanolic Acid (IC50 value = 13.45 ± 0.0 µM). The Kinetics mechanism for compound 4b was analyzed by Lineweaver-Burk plots which revealed that compound inhibited elastase competitively by forming an enzyme-inhibitor complex. Along with this, all the synthesized compounds (4a - 4j) exhibits excellent DPPH free radical scavenging ability. The inhibition constant Ki for compound 4b was found to be 0.6 µM. The computational study was comprehensible with the experimental results with good docking energy values (Kcal/mol). Therefore, these molecules can be considered as promising medicinal scaffolds for the treatment of skin-related maladies.
    Matched MeSH terms: Pancreatic Elastase/antagonists & inhibitors*; Pancreatic Elastase/metabolism; Pancreatic Elastase/chemistry
  4. Synthesis and structure-activity relationship of elastase inhibiting novel ethylated thiazole-triazole acetamide hybrids: Mechanistic insights through kinetics and computational contemplations
    Butt ARS, Abbasi MA, Aziz-Ur-Rehman, Siddiqui SZ, Hassan M, Raza H, et al.
    Bioorg Chem, 2019 05;86:197-209.
    PMID: 30711702 DOI: 10.1016/j.bioorg.2019.01.040
    Keeping in mind the pharmacological importance of 2-aminothiazole and 1,2,4-triazole heterocyclic moieties, a series of novel ethylated bi-heterocyclic acetamide hybrids, 9a-p, was synthesized in a multi-step protocol. The structures of newly synthesized compounds were characterized by 1H NMR, 13C NMR, IR and EI-MS spectral studies. The inhibitory effects of these bi-heterocyclic acetamides (9a-n) were evaluated against elastase and all these molecules were identified as potent inhibitors relative to the standard used. The Kinetics mechanism was analyzed by Lineweaver-Burk plots which revealed that, 9h, inhibited elastase competitively by forming an enzyme-inhibitor complex. The inhibition constants Ki calculated from Dixon plots for this compound was 0.9 µM. The computational study was articulate with the experimental results and these ligands unveiled good binding energy values (kcal/mol). So, these molecules can be considered as promising medicinal scaffolds for the treatment of skin melanoma, wrinkle formation, uneven pigmentation, and solar elastosis.
    Matched MeSH terms: Pancreatic Elastase/antagonists & inhibitors*; Pancreatic Elastase/metabolism
  5. Organic-solvent stability of elastase strain K overexpressed in an Escherichia-Pseudomonas expression system
    Wong CF, Salleh AB, Basri M, Abd Rahman RN
    Biotechnol Appl Biochem, 2010 Sep;57(1):1-7.
    PMID: 20726840 DOI: 10.1042/BA20100224
    The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.
    Matched MeSH terms: Pancreatic Elastase/genetics; Pancreatic Elastase/metabolism; Pancreatic Elastase/chemistry*
  6. Unravelling protein -organic solvent interaction of organic solvent tolerant elastase from Pseudomonas aeruginosa strain K crystal structure
    Said ZSAM, Arifi FAM, Salleh AB, Rahman RNZRA, Leow ATC, Latip W, et al.
    Int J Biol Macromol, 2019 Apr 15;127:575-584.
    PMID: 30658145 DOI: 10.1016/j.ijbiomac.2019.01.056
    The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- β-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.
    Matched MeSH terms: Pancreatic Elastase/chemistry*
  7. Fulltext Role of α-helical structure in organic solvent-activated homodimer of elastase strain K
    Rahman RN, Salleh AB, Basri M, Wong CF
    Int J Mol Sci, 2011;12(9):5797-814.
    PMID: 22016627 DOI: 10.3390/ijms12095797
    Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
    Matched MeSH terms: Pancreatic Elastase/genetics; Pancreatic Elastase/metabolism; Pancreatic Elastase/chemistry*
  8. Fulltext Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa
    Wong CF, Rahman RNZRA, Basri M, Salleh AB
    Iran J Biotechnol, 2017;15(3):194-200.
    PMID: 29845069 DOI: 10.15171/ijb.1524
    Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
    Matched MeSH terms: Pancreatic Elastase
  9. Fulltext A Marine Actinomycete Rescues Caenorhabditis elegans from Pseudomonas aeruginosa Infection through Restitution of Lysozyme 7
    Fatin SN, Boon-Khai T, Shu-Chien AC, Khairuddean M, Al-Ashraf Abdullah A
    Front Microbiol, 2017;8:2267.
    PMID: 29201023 DOI: 10.3389/fmicb.2017.02267
    The resistance of Pseudomonas aeruginosa to conventional antimicrobial treatment is a major scourge in healthcare. Therefore, it is crucial that novel potent anti-infectives are discovered. The aim of the present study is to screen marine actinomycetes for chemical entities capable of overcoming P. aeruginosa infection through mechanisms involving anti-virulence or host immunity activities. A total of 18 actinomycetes isolates were sampled from marine sediment of Songsong Island, Kedah, Malaysia. Upon confirming that the methanolic crude extract of these isolates do not display direct bactericidal activities, they were tested for capacity to rescue Caenorhabditis elegans infected with P. aeruginosa strain PA14. A hexane partition of the extract from one isolate, designated as Streptomyces sp. CCB-PSK207, could promote the survival of PA14 infected worms by more than 60%. Partial 16S sequence analysis on this isolate showed identity of 99.79% with Streptomyces sundarbansensis. This partition did not impair feeding behavior of C. elegans worms. Tested on PA14, the partition also did not affect bacterial growth or its ability to colonize host gut. The production of biofilm, protease, and pyocyanin in PA14 were uninterrupted, although there was an increase in elastase production. In lys-7::GFP worms, this partition was shown to induce the expression of lysozyme 7, an important innate immunity defense molecule that was repressed during PA14 infection. GC-MS analysis of the bioactive fraction of Streptomyces sp. CCB-PSK207 revealed the presence of methyl esters of branched saturated fatty acids. In conclusion, this is the first report of a marine actinomycete producing metabolites capable of rescuing C. elegans from PA14 through a lys-7 mediated activity.
    Matched MeSH terms: Pancreatic Elastase
  10. Fulltext NMR-Based Metabolomics Profiling for Radical Scavenging and Anti-Aging Properties of Selected Herbs
    Hussin M, Abdul Hamid A, Abas F, Ramli NS, Jaafar AH, Roowi S, et al.
    Molecules, 2019 Sep 03;24(17).
    PMID: 31484470 DOI: 10.3390/molecules24173208
    Herbs that are usually recognized as medicinal plants are well known for their therapeutic effects and are traditionally used to treat numerous diseases, including aging. This study aimed to evaluate the metabolite variations among six selected herbs namely Curcurmalonga, Oenanthejavanica, Vitex negundo, Plucheaindica, Cosmoscaudatus and Persicariaminus using proton nuclear magnetic resonance (1H-NMR) coupled with multivariate data analysis (MVDA). The free radical scavenging activity of the extract was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assay. The anti-aging property was characterized by anti-elastase and anti-collagenase inhibitory activities. The results revealed that P. minus showed the highest radical scavenging activities and anti-aging properties. The partial least squares (PLS) biplot indicated the presence of potent metabolites in P. minus such as quercetin, quercetin-3-O-rhamnoside (quercitrin), myricetin derivatives, catechin, isorhamnetin, astragalin and apigenin. It can be concluded that P. minus can be considered as a potential source for an anti-aging ingredient and also a good free radical eradicator. Therefore, P. minus could be used in future development in anti-aging researches and medicinal ingredient preparations.
    Matched MeSH terms: Pancreatic Elastase
  11. Proteomes of the female salivary glands of Simulium nigrogilvum and Simulium nodosum, the main human-biting black flies in Thailand
    Hempolchom C, Reamtong O, Sookrung N, Srisuka W, Sakolvaree Y, Chaicumpa W, et al.
    Acta Trop, 2019 Jun;194:82-88.
    PMID: 30922801 DOI: 10.1016/j.actatropica.2019.03.026
    Although several studies have reported pharmacological and immunological activity, as well as the role of black flies in transmitting pathogens to vertebrate hosts through salivary glands (SG) during blood feeding, SG proteomes of the anthropophilic black flies in Thailand have never been reported. Therefore, this study determined the SG proteomes of female S. nigrogilvum and S. nodosum. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2-DE) gels containing separated SG proteins of individual species were subjected to liquid chromatography-tandem mass spectrometry (LCMS/MS) and an orthologous protein search from eukaryotic organism, nematocera and simuliidae databases for total protein identification. SDS-PAGE and protein staining revealed at least 13 and 9 major protein bands in the SGs of female S. nigrogilvum and S. nodosum, respectively, as well as several minor ones. The 2-DE demonstrated a total of 56 and 41 protein spots for S. nigrogilvum and S. nodosum, respectively. Most of the proteins obtained in both species were enzymes involved in blood feeding, including proteases, apyrases, hyaluronidases, aminopeptidase and elastase. The results obtained in this study provided a new body of knowledge for a better understanding on the role of salivary gland proteins in these black fly species in Thailand.
    Matched MeSH terms: Pancreatic Elastase
  12. Fulltext Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol alleviates elastase-induced emphysema in a mouse model
    Chen YB, Lan YW, Chen LG, Huang TT, Choo KB, Cheng WT, et al.
    Cell Stress Chaperones, 2015 Nov;20(6):979-89.
    PMID: 26243699 DOI: 10.1007/s12192-015-0627-7
    Chronic obstructive pulmonary disease (COPD) is a sustained blockage of the airways due to lung inflammation occurring with chronic bronchitis and/or emphysema. Progression of emphysema may be slowed by vascular endothelial growth factor A (VEGFA), which reduces apoptotic tissue depletion. Previously, authors of the present report demonstrated that cis-resveratrol (c-RSV)-induced heat-shock protein 70 (HSP70) promoter-regulated VEGFA expression promoted neovascularization of genetically modified mesenchymal stem cells (HSP-VEGFA-MSC) in a mouse model of ischemic disease. Here, this same stem cell line was evaluated for its protective capacity to alleviate elastase-induced pulmonary emphysema in mice. Results of this study showed that c-RSV-treatment of HSP-VEGFA-MSC exhibited synergy between HSP70 transcription activity and induced expression of anti-oxidant-related genes when challenged by cigarette smoke extracts. Eight weeks after jugular vein injection of HSP-VEGFA-MSC into mice with elastase-induced pulmonary emphysema followed by c-RSV treatment to induce transgene expression, significant improvement was observed in respiratory functions. Expression of VEGFA, endogenous nuclear factor erythroid 2-related factor (Nrf 2), and manganese superoxide dismutase (MnSOD) was significantly increased in the lung tissues of the c-RSV-treated mice. Histopathologic examination of treated mice revealed gradual but significant abatement of emphysema and restoration of airspace volume. In conclusion, the present investigation demonstrates that c-RSV-regulated VEGFA expression in HSP-VEGFA-MSC significantly improved the therapeutic effects on the treatment of COPD in the mouse, possibly avoiding side effects associated with constitutive VEGFA expression.
    Matched MeSH terms: Pancreatic Elastase/pharmacology*
  13. Fulltext Predifferentiated amniotic fluid mesenchymal stem cells enhance lung alveolar epithelium regeneration and reverse elastase-induced pulmonary emphysema
    Lan YW, Yang JC, Yen CC, Huang TT, Chen YC, Chen HL, et al.
    Stem Cell Res Ther, 2019 06 13;10(1):163.
    PMID: 31196196 DOI: 10.1186/s13287-019-1282-1
    INTRODUCTION: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs).

    METHODS: Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration.

    RESULTS: An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups.

    CONCLUSION: Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice.

    Matched MeSH terms: Pancreatic Elastase/toxicity
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