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Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15

Yaacob MA, Hasan WA, Ali MS, Rahman RN, Salleh AB, Basri M, et al.

Acta Biochim. Pol., 2014;61(4):745-52.
PMID: 25337608

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.
Antimutagenic effects of aqueous fraction of Myristica fragrans (Houtt.) leaves on Salmonella typhimurium and Mus musculus

Akinboro A, Bin Mohamed K, Asmawi MZ, Yekeen TA

Acta Biochim. Pol., 2014;61(4):779-85.
PMID: 25520963

Natural plant extracts offer a promising hope in the prevention/treatment of cancer arising from genetic mutations. This study evaluated in vitro and in vivo mutagenic and antimutagenic effects of aqueous fraction of Myristica fragrans (AFMF) leaves on TA100 strain of Salmonella typhimurium and Mus musculus (Male Swiss albino mice), respectively. The antioxidant activity of AFMF against 2,2-diphenyl-1-picrylhydrazyl (DPPH), total phenolic and flavonoid contents were determined, followed by its phytochemical elucidation using the Ultra Performance Liquid Chromatography technique (UPLC). The mutagenicity of AFMF at 4, 20, 50, 100, 200, 500, and 1000 µg/well was <2.0 in S. typhimurium and the induced micronucleated polychromatic and normochromatic erythrocytes at 500, 1000, 2000, and 4000 mg/kg were not significantly different from the negative control (p≥0.05). The mutagenic activity of benzo[a]pyrene and cyclophosphamide was significantly suppressed above 50.0% throughout the tested concentrations. Fifty percent of the free radicals from DPPH were scavenged by AFMF at 0.11 mg/ml. Total phenolic and flavonoid contents of AFMF were 51.0 mg GAE/g and 27 mg QE/g, respectively. Rutin was elucidated by the UPLC technique, and thereby suspected to be the phytochemical responsible for the observed antimutagenic activity. Thus far, AFMF seems to contain a promising chemotherapeutic agent for the prevention of genetic damage that is crucial for cancer development.
Locally isolated yeasts from Malaysia: identification, phylogenetic study and characterization

Oslan SN, Salleh AB, Rahman RN, Basri M, Chor AL

Acta Biochim. Pol., 2012;59(2):225-9.
PMID: 22577620

Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 µg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 µg/mL and 100 µg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.
Hepatocyte nuclear factor 4 alpha P2 promoter variants associate with insulin resistance

Saif-Ali R, Harun R, Al-Jassabi S, Wan Ngah WZ

Acta Biochim. Pol., 2011;58(2):179-86.
PMID: 21633728

This study aimed to investigate the associations of hepatocyte nuclear factor 4 (HNF4) alpha single nucleotide polymorphisms (SNPs) and haplotype with insulin resistance and metabolic syndrome parameters. Nine SNPs spanning the HNF4 alpha P2 promoter (rs4810424, rs1884613 and rs1884614) and coding region (rs2144908, rs6031551, rs6031552, rs1885088, rs1028583 and rs3818247) were genotyped in 160 subjects without diabetes or metabolic syndrome. The HNF4 alpha P2 promoter SNPs rs4810424, rs1884613 and rs1884614 were associated with insulin resistance (p = 0.017; 0.037; 0.024) and body mass index (BMI) (p = 0.03; 0.035; 0.039). The intron 1D SNP rs2144908 was associated with high-density lipoprotein cholesterol (HDLc) (p = 0.020) and the intron 9 SNP rs3818247 showed association with systolic (p = 0.02) and diastolic (p = 0.034) blood pressure. HNF4 alpha common haplotype CCCGTC associated with higher insulin resistance (p = 0.022), fasting blood glucose (FBG) (p = 0.035) and lower HDLc (p = 0.001). In conclusion, subjects with HNF4 alpha P2 variants and haplotypes have been shown to have a higher insulin resistance and are therefore at a higher risk for developing type 2 diabetes mellitus.
Conformational destabilization of Bacillus licheniformis α-amylase induced by lysine modification and calcium depletion

Tan CY, Rahman RN, Kadir HA, Tayyab S

Acta Biochim. Pol., 2011;58(3):405-12.
PMID: 21887412

Bacillus licheniformis α-amylase (BLA) was chemically modified using 100-fold molar excess of succinic anhydride over protein or 0.66 M potassium cyanate to obtain 42 % succinylated and 81 % carbamylated BLAs. Size and charge homogeneity of modified preparations was established by Sephacryl S-200 HR gel chromatography and polyacrylamide gel electrophoresis. Conformational alteration in these preparations was evident by the larger Stokes radii (3.40 nm for carbamylated and 3.34 nm for succinylated BLAs) compared to 2.43 nm obtained for native BLA. Urea denaturation results using mean residue ellipticity (MRE) as a probe also showed conformational destabilization based on the early start of transition as well as ΔG(D)(H(2)O) values obtained for both modified derivatives and Ca-depleted BLA. Decrease in ΔG(D)(H(2)O) value from 5,930 cal/mol (for native BLA) to 3,957 cal/mol (for succinylated BLA), 3,336 cal/mol (for carbamylated BLA) and 3,430 cal/mol for Ca-depleted BLA suggested reduced conformational stability upon modification of amino groups of BLA or depletion of calcium. Since both succinylation and carbamylation reactions abolish the positive charge on amino groups (both α- and ε- amino), the decrease in conformational stability can be ascribed to the disruption of salt bridges present in the protein which might have released the intrinsic calcium from its binding site.
Fulltext Molecular cloning, sequence analysis and developmental stage expression of a putative septin gene fragment from Aedes albopictus (Diptera: Culicidae)

Avicor SW, Wajidi MF, Jaal Z, Yahaya ZS

Acta Biochim. Pol., 2016;63(2):243-6.
PMID: 27059016 DOI: 10.18388/abp.2014_909

Septins belong to GTPases that are involved in vital cellular activities, including cytokinesis. Although present in many organisms, they are yet to be isolated from Aedes albopictus. This study reports for the first time on a serendipitous isolation of a partial septin sequence from Ae. albopictus and its developmental expression profile. The Ae. albopictus partial septin sequence contains 591 nucleotides encoding 197 amino acids. It shares homology with several insect septin genes and has a close phylogenetic relationship with Aedes aegypti and Culex quinquefasciatus septins. The Ae. albopictus septin fragment was differentially expressed in the mosquito's developmental stages, with an increased expression in the adults.
Fulltext Optimization of culture conditions for flexirubin production by Chryseobacterium artocarpi CECT 8497 using response surface methodology

Venil CK, Zakaria ZA, Ahmad WA

Acta Biochim. Pol., 2015;62(2):185-90.
PMID: 25979288 DOI: 10.18388/abp.2014_870

Flexirubins are the unique type of bacterial pigments produced by the bacteria from the genus Chryseobacterium, which are used in the treatment of chronic skin disease, eczema etc. and may serve as a chemotaxonomic marker. Chryseobacterium artocarpi CECT 8497, an yellowish-orange pigment producing strain was investigated for maximum production of pigment by optimizing medium composition employing response surface methodology (RSM). Culture conditions affecting pigment production were optimized statistically in shake flask experiments. Lactose, l-tryptophan and KH2PO4 were the most significant variables affecting pigment production. Box Behnken design (BBD) and RSM analysis were adopted to investigate the interactions between variables and determine the optimal values for maximum pigment production. Evaluation of the experimental results signified that the optimum conditions for maximum production of pigment (521.64 mg/L) in 50 L bioreactor were lactose 11.25 g/L, l-tryptophan 6 g/L and KH2PO4 650 ppm. Production under optimized conditions increased to 7.23 fold comparing to its production prior to optimization. Results of this study showed that statistical optimization of medium composition and their interaction effects enable short listing of the significant factors influencing maximum pigment production from Chryseobacterium artocarpi CECT 8497. In addition, this is the first report optimizing the process parameters for flexirubin type pigment production from Chryseobacterium artocarpi CECT 8497.
Fulltext Metabolomics approach for multibiomarkers determination to investigate dengue virus infection in human patients

Shahfiza N, Osman H, Hock TT, Abdel-Hamid AZ

Acta Biochim. Pol., 2017;64(2):215-219.
PMID: 28350402 DOI: 10.18388/abp.2015_1224

BACKGROUND: Dengue is one of the major public health problems in the world, affecting more than fifty million cases in tropical and subtropical region every year. The metabolome, as pathophysiological end-points, provide significant understanding of the mechanism and progression of dengue pathogenesis via changes in the metabolite profile of infected patients. Recent developments in diagnostic technologies provide metabolomics for the early detection of infectious diseases.
METHODS: The mid-stream urine was collected from 96 patients diagnosed with dengue fever at Penang General Hospital (PGH) and 50 healthy volunteers. Urine samples were analyzed with proton nuclear magnetic resonance (1H NMR) spectroscopy, followed by chemometric multivariate analysis. NMR signals highlighted in the orthogonal partial least square-discriminant analysis (OPLS-DA) S-plots were selected and identified using Human Metabolome Database (HMDB) and Chenomx Profiler. A highly predictive model was constructed from urine profile of dengue infected patients versus healthy individuals with the total R2Y (cum) value 0.935, and the total Q2Y (cum) value 0.832.
RESULTS: Data showed that dengue infection is related to amino acid metabolism, tricarboxylic acid intermediates cycle and β-oxidation of fatty acids. Distinct variations in certain metabolites were recorded in infected patients including amino acids, various organic acids, betaine, valerylglycine, myo-inositol and glycine.
CONCLUSION: Metabolomics approach provides essential insight into host metabolic disturbances following dengue infection.
Fulltext Assessment of fracture healing properties of lovastatin loaded nanoparticles: preclinical study in rat model

Zhu P, Huang G, Zhang B, Zhang W, Dang M, Huang Z

Acta Biochim. Pol., 2019 Mar 11;66(1):71-76.
PMID: 30856636 DOI: 10.18388/abp.2018_2719

Bone fracture, being mainly caused by mechanical stress, requires special and quick attention for a rapid healing. The study presented here aims at formulating nanoparticulate system to overcome the solubility issues of lovastatin. The lovastatin nanoparticles were successfully prepared by ionotropic gelation method using chitosan and tri-polyphosphate as polymers. Thus prepared nanoparticles were found to be smooth and spherical with average particle size of 87 nm and encapsulation efficiency of 86.5%. The in-vitro drug release was found to be almost 89.6% in the first 360 minutes. Artificial fracture was produced in female Wistar rats at right leg using fracture apparatus. After administration of lovastatin nanoparticles or saline solution, the respective groups were observed for various parameters. The X-ray imaging showed that lovastatin accelerated bone healing, compared to control. The growth of animals was not hampered by lovastatin by any means. The radiographic examination confirmed a role of lovastatin in increasing bone density. The histological study showed the broken, proliferated and discontinued trabecullae in the control, while at the same time point, the normal, thick, continuous and connected trabecullae were observed in animals administered with lovastatin nanoparticles. The biomechanical studies showed high breaking resilience and minimum bone brittleness in animals injected with lovastatin nanoparticles. Considering these observations we state that lovastatin helps in rapid bone healing after fracture via increasing the bone density.
Fulltext In vitro alpha-glucosidase and alpha-amylase enzyme inhibitory effects of Andrographis paniculata extract and andrographolide

Subramanian R, Asmawi MZ, Sadikun A

Acta Biochim. Pol., 2008;55(2):391-8.
PMID: 18511986

There has been an enormous interest in the development of alternative medicines for type 2 diabetes, specifically screening for phytochemicals with the ability to delay or prevent glucose absorption. The goal of the present study was to provide in vitro evidence for potential inhibition of alpha-glucosidase and alpha-amylase enzymes, followed by a confirmatory in vivo study on rats to generate a stronger biochemical rationale for further studies on the ethanolic extract of Andrographis paniculata and andrographolide. The extract showed appreciable alpha-glucosidase inhibitory effect in a concentration-dependent manner (IC(50)=17.2+/-0.15 mg/ml) and a weak alpha-amylase inhibitory activity (IC(50)=50.9+/-0.17 mg/ml). Andrographolide demonstrated a similar (IC(50)=11.0+/-0.28 mg/ml) alpha-glucosidase and alpha-amylase inhibitory activity (IC(50)=11.3+/-0.29 mg/ml). The positive in vitro enzyme inhibition tests paved way for confirmatory in vivo studies. The in vivo studies demonstrated that A. paniculata extract significantly (P<0.05) reduced peak blood glucose and area under curve in diabetic rats when challenged with oral administration of starch and sucrose. Further, andrographolide also caused a significant (P<0.05) reduction in peak blood glucose and area under the curve in diabetic rats. Hence alpha-glucosidase inhibition may possibly be one of the mechanisms for the A. paniculata extract to exert antidiabetic activity and indicates that AP extract can be considered as a potential candidate for the management of type 2 diabetes mellitus.

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