April - June 2007 - Kasetsart University

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342 DISCUSSION The ability to produce and purify large quantities of biologically active interleukin-2 has been made possible by the use of recombinant DNA technology. The mouse cDNA library of BALB/c strain was cloned and characterized for <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) its activity. The IL-2 cDNA consisted of 450 base pairs, repeating CAG and showed high homology at 100%, 100%, 91%, 96% and 94% with five strains of mice previously reported (GeneBank accession number AY147902.1, MMU41494, MMU41504, MMU41505 and MMU41506). The rmIL-2 has been intensively studied and found that Figure 4 Detection of the recombinant mouse IL-2 (rmIL-2) by SDS-PAGE (A) and Western blotting (B) by probing with mouse IL-2 specific monoclonal antibody. M; molecular weight markers. Figure 5 The effect of various concentrations of the recombinant IL-2 on cell proliferation of the lymphocytes determined by XTT colorimetric assay while non-incubated cells with the recombinant IL-2 showed the OD450 nm = 0.248 (data not shown).
other strains of mice have different effects on the biological activity of IL-2 (Matesanz and Alcina, 1996). The IL-2 cDNA did not contain the hydrophobic leader sequence of a 20 amino acid peptide and the expressed rmIL-2 was purified from the cells later. According to Robb et al. (1981), even though the IL-2 exhibited O-linked glycosylation at threonine 3 of N-terminus and the E. coli system did not provide the posttranslational glycosylation, it did not affect the IL-2 activity nor change its activity in standard bioassay. The functional significance of glycosylation of IL-2 was not known but it was likely that it enhances solubility in aqueous environments. Thus an Nterminal 20 amino acid sequence was reported to be essential for the interaction with the IL-2 receptor (Eckenberg et al., 2000). The polymorphism of the CAG sequence has been reported among different strains of mice including C3HeB/FeJ mouse (AY147902.1), RF mouse (MMU41494), C57BL6/J mouse (MMU41504), CZECHII/Ei mouse (MMU41505), and BKL <strong>Kasetsart</strong> J. (Nat. Sci.) 41(2) 343 mouse (MMU41506) (GeneBank data base) which contained the sequences of 8, 8, 12, 21 and 21 codons, respectively. Characterization of the rmIL- 2 by ProtParam program (ExPASy) showed that it consisted of 149 amino acids of mature IL-2 protein and 12 amino acids of protein tag from the expression vector. The expressed protein was estimated to weigh 18,489 Da, with the isoelectric point (pI) at 5.87 with good solubility. However, the protein bands observed on the SDS-PAGE were found to be 19 and 38 kDa which were predicted as a monomeric and dimeric forms of the protein. The increased molecular weight from the data obtained by program analysis may due to the phosphorylation of the rmIL-2 (Adachi et al., 1997; Brennan et al., 1997; Gesbert et al., 1998; Justement, 2001; Cook and Unger, 2002; Michelle et al., 2003; Stoker, 2005). The rmIL-2 were applied to the cell culture with the following addition of XTT to examine its biological activity. The result showed that the activity was raised according to the increasing concentration of rmIL- Figure 6 The binding of the recombinant mouse IL-2 to the IL-2 receptors was analyzed by immunofluorescence. Cells stained with sulforhodamine B (SRB) illustrating red color and the signal for IL-2 binding showed greenish fluorescence (arrows). (A) Non-stimulated cells and (B) mitogen-stimulated cells, after 6 h of incubation.
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April - June 2007 Volume 41 Number
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KASETSART JOURNAL NATURAL SCIENCE T
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Kasetsart J. (Nat. Sci.) 41 : 205 -
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harvesting time which started from
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y Chen and Paull (2000). Figure 1 a
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pineapple fruit allowed the accumul
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Kasetsart J. (Nat. Sci.) 41(2) 215
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Cluster analysis Cluster analysis u
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Table 3 (Continued) 1 2 3 4 5 6 7 8
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CONCLUSION AFLP markers classified
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difference of soil texture used in
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under wet soil condition planting.
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tropics. Following the expansion, w
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sealed in a plastic box with 1 cm w
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moisture content increment after so
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Frequency Frequency 30 20 10 0 20.0
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School, Kasetsart University. The a
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for combining ability of lines (Lam
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selective mass sibbing within indiv
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Although most of S 2-interfamily hy
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composite sets as used in this stud
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days. The numbers of calli producin
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the report of Ching (1982) showing
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clearly amplified bands generated b
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caused by either the recombination
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Kuhlmann, V. and B. Foroughi-Wehr.
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Xanthomonas fuscans subsp. aurantif
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was collected and washed with 70% e
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expected size was amplified with 35
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eaction technique has been used for
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Schaad, N.W., D. Opgenorth and P. G
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MATERIALS AND METHODS Model descrip
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calculated TF value calculated TF v
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sincere gratitude and deep apprecia
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1989). In monogastrics, the inclusi
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of all LLS samples in this study we
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Melbourne, Parkville, Victoria. Goe
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considered responsible for low prod
Page 91 and 92: Kasetsart J. (Nat. Sci.) 41(2) 291
Page 93 and 94: mineral supplementation suggested b
Page 95 and 96: Body weight chane (kg/head) 32 30 2
Page 97 and 98: CONCLUSION AND RECOMMENDATION With
Page 99 and 100: SAS. (Statistical Analysis System).
Page 101 and 102: genes covering a variety of desirab
Page 103 and 104: mg/l NAA and 0.5 mg/l zeatin) incub
Page 105 and 106: 5. Purification by various sucrose
Page 107 and 108: as the culture period increased. So
Page 109 and 110: culture, pp. 1-20. In L.C. Fowke an
Page 111 and 112: Kasetsart J. (Nat. Sci.) 41 : 311 -
Page 113 and 114: GC. Analytical methods Total carboh
Page 115 and 116: ash. The crude hot water polysaccha
Page 117 and 118: spirulan (H-SP), and a desulfated c
Page 121 and 122: cells often displayed an increased
Page 123 and 124: LITERATURE CITED Bell, T.A. and D.V
Page 125 and 126: for alternative sources of supply.
Page 127 and 128: BR2.1.2 formed the same clade with
Page 129 and 130: supplement with glutamate (Iida et
Page 131 and 132: optimal for S. limacinum BR2.1.2 fo
Page 133 and 134: fluorescent lamp at 1.5 kLux develo
Page 137 and 138: Wisconsin, USA). Total extracted RN
Page 139 and 140: RESULTS Cloning and sequencing of m
Page 141: Expression of rmIL-2 and purificati
Page 145 and 146: dose interleukin-2 therapy promotes
Page 147 and 148: The chitosano-oligosaccharides are
Page 149 and 150: enzyme and cells were maximized by
Page 151 and 152: of crude chitosanases. Fortunately,
Page 153 and 154: Relative activity (%) 100 80 60 40
Page 155 and 156: Uchida, K. Tateishi, O. Shida and K
Page 157 and 158: MATERIALS AND METHODS 1. Materials
Page 159 and 160: without coating and coated with pec
Page 161 and 162: in Table 5-7. It was found that the
Page 165 and 166: the cultures at 10,200 × g for 15
Page 167 and 168: incubation and reached the final pH
Page 169 and 170: y lysine- and tyrosine-decarboxylas
Page 171 and 172: during the stages of ripening as su
Page 175 and 176: As the 25 companies were divided in
Page 177 and 178: All the data comes up with informat
Page 179 and 180: as piece “A” or straight as pie
Page 181 and 182: et al. (1998); Zeng (2000); and Tal
Page 183 and 184: p 1, i * Kasetsart J. (Nat. Sci.) 4
Page 185 and 186: c ≤ c2 jq2 j n q p k p * 2, j , ,
Page 187 and 188: algorithm would consider RM 5, prio
Page 189 and 190: Kasetsart J. (Nat. Sci.) 41(2) 389
Page 191 and 192: the maximum deviation of about 10%.
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Stock Quantities Using Heuristic Op
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which dynamically and automatically
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2. Framework architecture and compo
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Risk (VaR) calculation which was im
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Throughput (job/sec) 7 6 5 4 3 2 1
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Speed up 40 35 30 25 20 15 10 5 0 F
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and discovery, resource selection a
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April - June 2007 - Kasetsart University

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