Pengaruh Superoksida Dismutase Rekombinan Staphylococcus equorum Terhadap Viabilitas Sel dan Deposisi Kolagen Pada Sel Fibroblas 3T3 Yang Dipaparkan UVA

UVA is the main external factors causing skin aging. UVA increase matrix metalloproteinase (MMP) synthesis that degrade collagen indirectly through reactive oxygen species. Superoksida dismutase (SOD) catalyzes the conversion of superoxide anions to hydrogen peroxide and oxygen. SOD can be used as a cosmetic’s component to prevent skin aging. The objective of this research to determine rSOD S. equorum ability to increase collagen deposition using fibroblast 3T3 against UVA. The research was initiated by the confirmation of E. coli BL21(DE3) carrying pJExpress®414-sod. rSOD was overproduced in E. coli BL21(DE3) by IPTG induction to a final concentration of 1 mM for 4 hours at 37oC. rSOD purification was done using an Ni-NTA affinity column with gradient imidazole concentrations for elution. Various concentrations of rSOD was added to fibroblast 3T3 cell culture after UVA irradiation to determine its role in collagen deposition. The effect of rSOD was analyzed by fibroblast viability using Alamar blue and collagen deposition with picro sirius red. The results showed that fibroblast cell viability exposed with UVA for 45 minutes in the presence of 4, 8, and 16 unit rSOD was not significantly affected, however, the collagen deposition was significantly increased about 3.02%; 20.03% and 35.75% respectively compared to cell without rSOD. This result indicates that rSOD is a good candidate for an anti photoaging agent.UVA merupakan faktor eksternal utama penyebab penuaan kulit. UVA meningkatkan produksi matrik metalloproteinase (MMP) yang mendegradasi kolagen tidak langsung melalui pembentukan reactive oxygen species. Superoksida dismutase (SOD) mengkatalisis perubahan anion superoksida menjadi hidrogen peroksida dan oksigen. SOD dapat digunakan sebagai komponen kosmetik untuk mencegah penuaan kulit. Tujuan penelitian ini adalah, mengetahui pengaruh rSOD S. equorum dalam meningkatkan deposisi kolagen pada sel fibroblas 3T3 yangdipaparkan UVA. Penelitian diawali dengan konfirmasi E. coli BL21(DE3) yang membawa pJExpress®414- sod. Overproduksi rSOD dilakukan dengan IPTG hingga konsentrasi akhir 1 mM selama 4 jam pada suhu 37oC. Pemurnian rSOD dilakukan menggunakan kromatografi afinitas kolom nikel dengan gradien konsentrasi imidazol untuk elusi. Viabilitas sel ditentukan menggunakan Alamar blue, sedangkan deposisi kolagen dengan Sirius red. Hasil uji menunjukkan bahwa, paparan UVA selama 45 menit pada sel dengan 4, 8, dan 16 unit rSOD tidak mempengaruhi viabilitas sel secara bermakna. Namun, mampu meningkatkan deposisi kolagen berturut-turut sebesar 3,02%; 20,03% dan 35,75% dibandingkan dengan sel tanpa rSOD. Hasil ini menunjukkan bahwa rSOD mempunyai potensi sebagai agen anti fotoaging

Karakterisasi Molekular Fragmen Gen mexB Isolat Pseudomonas aeruginosa Multiresisten

Antibiotics have been widely used in the treatment of infectious diseases. However, their effectiveness has been questioned due to the tendency of some bacterial resistance to antibiotics. Pseudomonas aeruginosa among others has been known to be resistant to several antibiotics due to its MexABOprM efflux pump. Perhaps, the nucleotide sequence of its mexB gene fragment has changed followed by changes in amino acid sequence leading to alteration of the substrate recognition site. This alteration causes disability of antibiotics to recognize it and they are pumped out from the bacterial cell causing decrease in its inhibition concentration. An observasional study was performed using four P. aeruginosa isolates (A,B,C and D) taken from four laboratories in Bandung, and the sensitivity test for several antibiotics (tetracyclin, ampicyllin, amoxicyllin-clavulanat, kanamycin, ciprofloxazin, trimetoprim-sulphametoxazol, chloramphenicol dan eritromycin), was performed using Kirby-Bauer method. The Minimum Inhibitory Concentrations (MICs) for 4 isolates were 20.57-39.07 mg/ml for erithromycin, 29.35-48.57 mg/ml for kanamycin, 30.35-68.75 mg/ml for tetracyclin, 45.57-97.50 mg/ml for ampicyllin, 23.69-97.50 mg/ml for chloramphenicol, 25.82-59.56 mg/ml for amoxcyllinclavulanat,21.88-79.00 mg/ml for trimetoprim sulphametoxazol, and 20.58-56.97 mg/ml for ciprofloxazin. The increasing of MIC to each antibiotic was shown for 4 isolates of P. aeruginosa multiresistant to several antibiotics being studied. PCR technique was used to detect mexB gene fragment asumed as the substrate recognition site. The percentage of homology between the nucleotide sequence of mexB multiresistant P. aeruginosa and mexB P. aeruginosa producing siderophore pioverdin (Acc. No. L11616, NCBI) showed 96%, 100%, 97%, and 96% homology for P. aeruginosa A,B,C and D respectively. Employing DNAstar program, fragment variant of mexB gene of 4 multiresistant isolates A, B, C and D was detected. This variation lead to amino acid substitution of Gly-417-Ser, Glu-417-Gln, Thr-424-Pro, Tyr-328-Phe, Asp-328-His for P. aeruginosa A,B,C and D respectively, along with the change of their secondary structure, that changed the functional protein of MexB

Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction

Protein serial biokimia mudah dan menggugah

xviii, 250 hlm.: 23 c

STUDY ON THE PROPERTIES OF PURIFIED RECOMBINANT SUPEROXIDE DISMUTASE FROM STAPHYLOCOCCUS EQUORUM, A LOCAL ISOLATE FROM INDONESIA

Objective: Superoxide dismutase (SOD) (E. C: 1.15.1.1) from Staphylococcus equorum which catalyzes the dismutation of the superoxide anion (O2.-) into molecular oxygen (O2) and hydrogen peroxide (H2O2), is one of the most important classes of antioxidant enzymes and are used in pharmaceutical or cosmetic applications. SOD of S. equorum was purified from total protein into homogeneity and characterized to determine the unit activity, ion metal cofactor, optimum temperature and pH, kinetic parameters, and effect of denaturing and reducing agents and UVC exposure on the rSOD activity. Methods: The protein was purified in a single-step purification using Ni-NTA afï¬nity column with various imidazole concentrations. SOD activity was analyzed by colorimetric and activity staining using nitroblue tetrazolium (NBT). The purified rSOD was exposed to different temperatures and pHs, different concentrations of denaturing agents, reducing agents, and to UVC exposure. Results: SOD protein with high purity was obtained when imidazole concentrations of 100 mM, 200 mM and 250 mM were applied. The purified rSOD displayed specific activity of 1666.7 U mg-1when measured at 30ºC and pH 7.8. The presence of conserved manganese-binding sites (H28, H83, D171, H175) and the inhibition of rSOD activity by NaN3 but not by H2O2 or KCN and indicated that rSOD was Mn-dependent. The optimum temperature and pH were determined to be 40ºC and 6.0, respectively. The Michaelis constant (Km), maximum velocity (Vmax), turnover number (kcat) and catalytic efficiency (kcat/Km) were found to be 371.2 µM, 1.738 µMS-1, 1.358 s-1, and 3.7x10-3 S-1µM-1, respectively. The rSOD activity was slightly affected in the presence of detergents (0.5% SDS, 0.5% Triton-X 100), denaturing agents (6 M GdnHCl and 6 M urea) and reducing agent (5 mM βME). After exposure of rSOD by UVC for 45 min, it retained half of its activity. Conclusion: This is the first study to report the stability of the SOD of S. equorum against environmental factors. The SOD displays some thermostability, is active in wide pH, stable in the presence of denaturing and reducing agents, however it is relatively unstable to UVC exposur

16S rDNA-Based Identification of Novel Superoxide Dismutase Producing Bacteria Isolated from Indonesia

Superoxide dismutase (SOD) has therapeutic importance because of its antioxidant activity and protects cells from reactive oxygen species attack. This research was intended to screen bacteria isolated from Indonesia for producing novel SODs and to identify the producers using 16S rDNA approach. Intracellular proteins were each extracted and assayed for their inhibition reduction activity by colorimetric method and by zymography for the presence of SOD protein band(s). For species identification, each of 16S rDNAgenes was amplified by polymerase chain reaction from genomicDNAfollowed by sequencing, BLAST, multiple alignment and phylogenetic analyses. All 16 intracellular proteins gave inhibition reduction percentage in the range of 15 to 70% and in zymography, their SOD profiles were quite diversed with at least one intenseSOD band present in most isolates. The SOD producers were assigned to three species, Flavobacterium okeanokoites, Escherichia fergunosii, and E. coli, and to four genera, Pantoea, Escherichia,  Bacillus, and Pectobacterium. The remaining five were grouped in gamma-proteobacterium cluster and two formed a cluster with Pseudomonas. Three marine and four soil isolates could be attractive candidates for novel SODs based on unique properties of SOD producers. In conclusion, 16s rDNA-based identification of bacteria isolated from Indonesia reveals that seven isolates might be attractive candidates for novel SOD producers to be applied in pharmaceutical fields in the future

Gliadin Peptide Facilitates FITC Dextran Transport across the Non Everted Gut Sac of Rat Small Intestine

Superoxide dismutase (SOD) is an antioxidant protein. When administered orally, it has low bioavailability due to its low permeation. In a previous study we fused gliadin peptide P51 (LGQQQPFPPQQPYPQPQPF) and gliadin peptide P61 (QQPYPQPQPF) with SOD Citrus limon (SOD_Cl), namely GliSOD_P51 and GliSOD_P61 to increase permeation of SOD_Cl through intestine. In this work, the permeation of fluorescein isothiocyanate (FITC)-Dextran 10 kDa, FD10 and 40 kDa, FD40 as paracellular transport markers across excised rat intestinal wall was investigated with the presence of GliSOD_P51 and GliSOD_P61. A permeability study was performed using non-everted rat intestine by incubating FD10 or FD40 with SOD_Cl, and GliSOD_P61. The presence of SOD_Cl, GliSOD_P51 or GliSOD_P61 inside intestine (apical) and outside intestine (basolateral) was analyzed by protein electrophoresis. The concentration of FD that penetrated to the basolateral solution was analyzed by spectrofluorometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the presence of GliSOD_P51 and GliSOD_P61 but not SOD_Cl in basolateral compartment. The percentage of FD10 but not FD40 and SOD_Cl that penetrated to the basolateral solution significantly increased with the presence of gliadin in GliSOD_P51 and GliSOD_P61. GliSOD_P51 and GliSOD_P61 are able to penetrate the rat intestinal epithelial membrane and the gliadin peptides facilitate FD10 to penetrate the epithelial

The Role of the First 14 Amino Acids of Mature M1 Protein of Streptococcus pyogenes on Fibronectin-Binding Activity and Dimer Formation

Streptococcus pyogenes is one of the most important human pathogens which express a multi-facet of virulence factors on its cell surface.  One of the virulence factors that has been intensively-studied is the M protein that binds several human proteins.  M1 protein, a member of the M protein family, was previously found to bind human fibronectin (Fn), an activity that is responsible for bacterial internalization. A structural study showed that this protein consists of four regions: A, B, S, and C.  The study  was intended to investigate the role of the first 14 amino acid residues located at the non-helical region of M1 protein in binding Fn, and its ability to form a dimer. The DNA fragment encoding for the ABS protein lacking its first 14 amino acids (ABSD14aa) was cloned into pET-16b, overexpressed in Escherichia coli BL21(DE3), and the protein was purified by affinity chromatography. The purified protein was characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis and the Fn-binding activtiy was assayed by enzyme linked immunosorbent assay.  The result indicated that the M1 lacking its first 14 amino acids retains its dimerization and Fn-binding activities

Protein: serial biokimia mudah dan menggugah

xviii, 250 hlm. : ilus. ; tab. ; 23 cm

T118N Substitution of Hepatitis B X Protein Reduces Colony Formation of HepG2 Cells

BACKGROUND: The acute Hepatitis B virus (HBV) infection usually ceases before six months, but chronic infection that lasts for more than six months might develop into liver cirrhosis and hepatocellular carcinoma (HCC). Viral particle load, HBV genotypes and association to the HBV x (HBx) gene mutations are the probable factors related to HCC occurrence. The mutation which leads to HBx T118N was found as the second most common HBx mutation in Indonesia, as compared to the known cancer-related HBx K130M/V131I mutant. However, the effect of T118N mutation and its combination with K130M/V131I on human hepatoma cells has not been elucidated well. Hence, this study was conducted to dissect the role of HBx T118N and its mutant combination in colony formation, as compared to the wild type HBx and cancer-related HBx K130M/V131I.METHODS: In this study, the genes encoding wild type HBx, HBx T118N, and HBx K130M/V131I mutations were obtained as synthetic gene. Meanwhile, the gene encoding HBx T118N/K130M/V131I mutations was successfully generated using site-directed mutagenesis. The optimum condition for colony formation assays was determined through Zeocin sensitivity test of HepG2 cells.RESULTS: Selection of HepG2 cells using Zeocin was determined at 200 µg/mL. Colony formation assays performed upon expression of HBx T118N and HBx T118N/K130M/V131I mutant proteins showed reduced colony numbers as compared to the expression of wild type HBx, similar to the effect from HBx K130M/V131I mutant expression.CONCLUSION: The HBx T118N and HBx T118N/K130M/V131I mutation caused less colony formation of HepG2 cells, similar to the K130/M131I mutation. This indicates a possible role of the T118N mutation in liver cancer development.KEYWORDS: colony formation assay, hepatitis B virus, HBx, T118N, K130M/V131