Indonesian Journal of Biotechnology
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Adherence Pheno-genotypic of Escherichia coli O157:H7 Isolated from Beef, Feces of Cattle, Chicken and Human
No full textGenerally, adherence of micro-organisms to host cells is the frst step of the colonization to host surfaces.Escherichia coli O157:H7 can colonize to the intestine and induce attaching-effacing (AE) lessions. The capacityof inducing AE lesions is encoded by a pahtogenicity island, the locus of enterocyte effacement (LEE) thatcontains genes involved in generation of attaching and effaching (A/E) lesions. Among which that, the eaegene is encoding intimin, an outer membrane protein that is responsible to intimate attachment to the intestinalepithelial cells. A total of 20 local isolates obtained from human clinically, beef, cattle, chicken, and humannon-clinically were tested to adherence pheno-genotypic of E. coli O157:H7. The eae gene was identifed usingpolymerase chain reaction with a specifc primer i.e AE19 forward and AE20 reverse. To confrm phenotypicof gene, further study was performed by culturing the bacteria in vero cell, followed by Giemsa staining andAcridine Orange Fluorescent staining 3 h and 6 h after incubation, respectivelly. Result of study showed thatthere were 19 out of 20 (95%) isolates identifed positive eae gene. Giemsa staining appeared that the bacteriawith positive eae gene performed a cluster arroud of cell (localized adherence). On the other hand, the negativeeae gene appeared as a diffuse adherence (DA). The study indicated that almost all of E. coli O157:H7 localisolates which was positive eae gene had potency to colonize to the intestine and induce attching-effachinglessions, and also cause cytopahatic effects in intestinal epithelial cel
Antigen Presentation Ability of Salmonella Carrying DNA Vaccine Model and MCP-3 gene
No full textThe objective of this study is to determine the antigen presentation ability of a DNA vaccine model that is co-delivered with that of recombinant Salmonella enterica serovar Typhimurium (STM1) expressing chemokine macrophage chemotactic protein-3 (MCP-3). The DNA vaccine, pVROVA, was constructed by amplification of the ovalbumin coding region from sOVA-C1. Dendritic cells (DCs) were obtained from IL-4 and GMCSF stimulated mouse bone marrow stem cell. Cultured DCs were incubated with STM1 carrying a model ovalbumin gene (pVROVA). Furthermore, MHC class I antigen presentation of a dominant OVA peptide was assayed in vitro. The experiments were designed to determine the effect of co-delivering MCP-3 with that of ovalbumin in STM1. Our results show that a plasmid pROVA-carrying ovalbumin gene was succesfully constructed and sequence analysis of the ovalbumin-coding revealed an identity match of 100% with that of the chicken ovalbumin DNA sequences from the GenBank database. We also found that the presence of the MCP-3 encoding plasmid in STM1 or E. coli DH1 could increase the recovery of both STM1 and E. coli DH1 over those that carry the empty plasmids. Antigen presentation assay also indicates that MCP-3 can positively influence the presentation of ovalbumin. Conclusion: the infection of DCs by STM1-carrying DNA vaccine and MCP-3 results in an increase of processing and presentation of ovalbumin in vitro.Keywords : DNA vaccine, MCP-3, APC, Salmonella, Dendritic cell
Antimicrobial and Cytotoxic Potential of the Compound Secreted by Marine Bacteria Collected from the Karwar Coast, West Coast of India
No full textAntibacterial substances from sea sediment were isolated from marine environment of Karrwar, westcoast of India and characterized. Out of the 21 isolates subjected to secondary screening, 10 isolates were activeagainst Bacillus subtilis, 12 against Staphylococcus aureus, 6 against Escherichia coli, 3 against Proteus vulgaris and4 against Salmonella typhi. Metabolites in the broth extract of overnight grown Pseudomonas spp. No.7 provedto have antimicrobial and cytotoxic against human lung carcinoma cel
DIGoxigenin (DIG) Labeled Probe Candidate of Surface Antigen 1 (SAG1) for Toxoplasma gondii Detection
No full textToxoplasma gondii is one of the opportunistic pathogen that causes toxoplasmosis. Infection of Toxoplasma gondii has been estimated as high both in human and animal. The manifestation of infection were abortion, hydrocephalus, brain calcification, chorioretinal scar, and loss of productivity even to death in patients with acquired immunosuppression. Early diagnostic method which are rapid and accurate is essential for T. gondii detection because of its high prevalence. The purpose of this study was to develop a sensitive probes derived from Surface Antigen 1 (SAG1) for detection T. gondii and to examine the specificity and sensitivity of probe as diagnostic tool for toxoplasmosis. This research used SAG1 gene of T. gondii local isolate IS-1 that was cloned into pGEX-2T and transformed into Eschericia coli DH5α. The sequence of SAG1 was labeled with DIGoxigenin (non radioactive labeled) using PCR DIG Labeling Mix to derive 213 bp (probe-TS). BLAST and dot-blot hybridization analyses showed that probes had high specifity with other strains of T. gondii. Probe was able to detect T. gondii DNAup to 10 ng/μl of total sample DNA
Molecular Identifcation of Lactic Acid Bacteria Producing Antimicrobial Agents from Bakasang, An Indonesian Traditional Fermented Fish Product
No full textTwenty seven strains of lactic acid bacteria (LAB) were isolated from bakasang, Indonesian traditionalfermented fsh product. In general, LAB have inhibitory activity againts pathogenic bacteria and spoilagebacteria. Screening for antimicrobia activity of isolates were performed with well-diffusion method. Oneisolate that was designed as Pediococcus BksC24 was the strongest against bacteria pathogenic and spoilagebacteria. This strain was further identifed by 16S rRNA gen sequence comparison. Isolates LAB producingantimicrobial agents from bakasang were identifed as Pediococcus acidilactici
T-786c Polymorphism in nitric oxide synthase 3 gene and Nitrit Oxide Level of Diabetic Retinopathy in Javanese Population
No full textComplication of retinopathy in type 2 DM is caused of lower level of NO. Nitric oxide level is synthesizedfrom L-arginin in reaction that catalyze Nitric oxide synthase (NOS) 3. The T-786C mutation in NOS 3 genedecreases the expression of nitric oxide synthase (NOS) 3 so decreases NO synthesis. To investigate theassociation between T-786C polymorphism in NOS 3 gene with NO level of diabetic retinopathy patients. Thisstudy was a case control study, consist of 40 patient of type 2 diabetic with DR (case group) and 40 patient oftype 2 diabetic without DR (control group) of Javanese ethnic. The genotyping of T-786C polymorphism wasperformed by PCR-RLFP. Level of NO was measured by spectrophotometry. Chi square test and odd ratiowere used to analyze the association of the T-786C polymorphism in NOS 3 gene with DR. Differences ofNO level between TT and TC genotypes were analyzed using independent t test. The distribution of T-786Cpolymorphism in NOS 3 gene of DR subjects showed that frequency of TT genotype was 22.5% and TC genotypewas 77.5%. Non DR subjects showed the frequency of TT genotype was 50% and TC genotype was 50%, (p=0.011). Frequency of T allele in DR group was 61.25% and C allele was 38.75%, and frequency of T allele in nonDR group was 75% and C allele was 25%, (p= 0.62). Odd ratio of TC genotype was 3.444(CI; 95% : 0.964-3.735)and C allele was 1.898 (CI; 95% : 1.310-9.058). The NO level of TC genotype was 1.43+0.126 and TT genotypewas 11.27+5.87 (p=0.000). Level of NO between RD and non RD showed not different significantly (p=0.160)for retinopathy. The T-786C polymorphism of NOS 3 gene is risk factor for retinopathy in type 2 DiabetesMellitus. Individual with TC genotype of NOS 3 gene has lower level of NO than TT genotype
Development of Random Amplified Polymorphism DNA Markers Linked to Powdery Mildew Resistance Gene in Melon
No full textA random amplified polymorphic DNA (RAPD) marker linked to powdery mildew resistance gene (Pm-I) in melon PI 371795 was reported. However, the RAPD marker has problem in scoring. To detect powdery mildew resistance gene (Pm-I) in melon accurately, the RAPD marker was cloned and sequenced to design sequence characterized amplified region (SCAR) markers. SCAPMAR5 marker derived from pUBC411 primer yielded a single DNA band at 1061 bp. Segregation of SCAPMAR5 marker in bulk of F2 plants demonstrated that the marker was co-segregated with RAPD marker from which the SCAR marker was originated. Moreover, results of SCAR analysis in diverse melons showed SCAPMAR5 primers obtained a single 1061 bp linked to Pm-I in resistant melon PI 371795 and PMAR5. On the other hand, SCAPMAR5 failed to detect Pm-I in susceptible melons. Results of this study revealed that SCAR analysis not only confirmed melons that had been clearly scored for resistance to Pm-I evaluated by RAPD markers, but also clarified the ambiguous resistance results obtained by the RAPD markers. Key words: Cucumis melo L., Pm-I, RAPD, SCAPMAR
CYP3A4*1G gene Polymorphism on Javanese People
No full textMost of drugs are metabolized by cytochrome P 450 (CYP) enzyme. Cytochrome P450 3A4 is thecytochrome that is involved in metabolizing more than 60% of all medicine used in human. The variationof this CYP3A4 gene will affect the catalytic activity of this enzyme. Recently, CYP3A4*1G in intron 10 wasfound in Chinese and Japanese population. There is a substitution of G to A at position 82266 in intron 10. Thepurpose of this research was to investigate the frequency of allele and genotype CYP3A4*1G. Samples weretaken from bloods of the subjects of the research. The examination of CYP3A4*1G was conducted by RTLP-PCRmethod.As the results of this research, the frequency of CYP3A4*1G in Javanese people is CYP3A4*1/*1 0.25,CYP3A4*1/*1G 0.55 and CYP3A4*1G/*1G 0.20. Frequency of allele G: 0.53, allele A: 0.47. The Fisher’s exact- testshows that the allele and genotype frequencyis p. 1.000. The allele and genotype frequency of Javanese peopleisstill in Hardy-Weinberg equilibrium
Application of Multiplex RT-PCR for Detection of Cucurbit-infecting Tobamovirus
No full textCucumber green mottle mosaic virus (CGMMV) and Kyuri green mottle mosaic virus (KGMMV) are seed borne viruses and they are also transmitted mechanically during agricultural practice and through water. Hence, these viruses have potential diseases widely distributed throughout the world. To detect different strains of CGMMV and KGMMV, several specific primers for each virus were designed for single and multiplex RT-PCR. The results of single and multiplex RT-PCR showed that CGMMV was detected in zucchini isolated in Bali-Indonesia, while KGMMV was detected both in zucchini isolated in Bali-Indonesia and Cucumis metuliferus isolated in Thailand. Furthermore, artificial co-infection of these two viruses was prepared and carried out using two different ways of viral RNAs extraction. Based on the results, it could be reported that viral RNAs for cDNA amplification by multiplex RT-PCR could be extracted from a mixture of infected leaves or separate extraction of each viruses infected leaves. In addition, results presented in this study demonstrated the application of multiplex RT-PCR to simultaneously detect CGMMV and KGMMV from cucurbit leaves using a mixture of four primers and its feasibility as a sensitive and rapid laboratory assay. Since, no multiplex RT-PCR technique has been described for the detection of CGMMV and KGMMV, this technique can be a good option for sensitive and reliable tool for detection of two major cucurbit infecting Tobamoviruses.Keywords : Cucurbit infecting Tobamovirus, multiplex RT-PCR, seed borne viruse
Ability of Curcuminoid from Curcuma domestica Val. in Reducing the Secretion of Reactive Oxygen Intermediates by Synovial Fluid Monocytes in Patients with Osteoarthritis
No full textIncreasing the secretion of reactive oxygen intermediates (ROI) by monocytes in the synovial fluid is anindicator to determine the severity of joint inflammation. Previous studies have shown that curcumin inhibitthe osteoarthritis progression with its ability to inhibite the activity of the nitric oxide synthase (NOS) enzymefrom macrophages. In this prospective randomized open end blinded evaluations = PROBE study, 80 patientswith knee osteoarthritis were eligable. The subject were devided in to two group: group who received 3 x 30mg of curcuminoid from Curcuma domestica Val. extract (curcuminoid group) and group who received 3 x 25mg of diclofenac sodium (diclofenac group) as comparison. The treatment was for 4 weeks time. The secretionof ROI by sinovial fluid monocytes was calculated by scoring the amount of formazan formation after neutralred staining in nitrobleu tetrazolium reduction assay. The result of this study showed that the secretion of ROIby synovial fluid monocytes was significantly decreased in both groups (p <0.001) respectively. There wasno significant difference in decreasing of ROI secretion of synovial fluid monocytes between both treatmentgroups (p = 0.92)