Hsien Jung Chen

Molecular cloning of two metallothionein-like protein genes with differential expression patterns from sweet potato (Ipomoea batatas) leaves

Metallothionein (MT) is a group of proteins with low molecular masses and high cysteine contents, and is classified into different types, which in general contains two domains (domain 1 and domain 2) with typical amino acid sequences (Rauser 1999). In this report two full-length cDNAs (Y459 and G14) encoding MT-like proteins were isolated from leaves of sweet potato (Ipomoea batatas). Their open reading frames contained 249 and 195 nucleotides (82 and 64 amino acids) for Y459 and G14, respectively, and exhibited a relatively low amino acid sequence similarity (ca. 25.8%). Gene structure studies showed that Y459 had the conserved domain 1 region of type 2 MT; however, the domain 2 region was not conserved and contained additional amino acids between the CxC and CxC spacing. G14 had conserved domains 1 and 2 of type 4 MT except that the last CxC of domain 2 was changed to RxC. Semi-quantitative RT-PCR showed that Y459 was expressed in significant quantity in roots and stems, but was much less in green leaves. During natural and induced (with dark and ethephon, an ethylene-releasing compound, treatments) leaf senescence, Y459 gene expression was significantly enhanced. In contrast, relatively constant gene expression levels were found for G14 in all tissues or treatments analyzed. In conclusion, the two MT-like protein genes of sweet potato display differential gene structures and gene expression patterns, which may be associated with the diverse roles and functions they play in plant physiology in order to cope with particular developmental and environmental cues.

Activity staining of glutathione peroxidase after electrophoresis on native and sodium dodecyl sulfate polyacrylamide gels

Glutathione peroxidase (GSH‐Px), from commercial bovine erythrocytes or ammonium sulfate fractionations (30–45%, 45–60%, 60–75% and 75–90% saturations) of ginger rhizome, was detected on polyacrylamide gels after native polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate (SDS)‐PAGE. The gel was submerged in a 50 mM Tris‐HCl buffer (pH 7.9) containing 13 mM glutathione and 0.004% hydrogen peroxide with gentle shaking for 10–20 min. The GSH‐Px activity was stained with a solution containing 1.2 mM 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 10 min. The clear zone of GSH‐Px activity on a purple background was found in both native and SDS‐PAGE gels. This fast and sensitive method can be used in the process of enzyme purification and characterization of mammalian or plant cells.

Antioxidant activities of methanolic and hot-water extracts from leaves of three cultivars of Mai-Men-Dong (Liriope spicata L.)

1,1-dipheny-2-picrylhydrazyl (DPPH) scavenging activities of the 80% methanolic leaf extracts of three cultivars (small leaf, SL; big leaf, BL; thin leaf, TL) of Mai-Men-Dong (Liriope spicata L.) are analyzed by spectrophotometry. The concentrations required for 50% inhibition (IC50) of DPPH radicals were 81.08, 96.97, and 53.78μg/mL, respectively. The methanolic extracts were further partitioned into three n-hexane-, ethylacetate-, and water-soluble fractions, among which the ethylacetate-soluble fraction exhibited the highest DPPH scavenging activity. The IC50 of ethylacetate-soluble fractions of SL, BL, and TL for DPPH radical scavenging activity were 41.55, 24.55, and 53.33μg/mL, respectively. Each Mai-Men-Dong powder (1g) was deposited in a tea bag and then dipped in hot water (100℃, 100mL) for 3 min with triplicate samples. These hot-water extracts were then freeze-dried for an anti-DPPH radical capacity test, which found a positive correlation with the phenolic contents of each hot water extract. The IC50 of hot water extracts of SL, BL, and TL for DPPH radical scavenging activities were 378.97, 171.12, and 95.84mg/mL, respectively. All three hot water extracts can effectively scavenge hydroxyl radical using electron spin resonance (ESR) spectrometry. The IC50 against hydroxyl radical were 80.8, 69.7, and 116μg/mL, respectively, for the SL, BL, and TL cultivars.

Activity staining of isocitrate lyase after electrophoresis on either native or sodium dodecyl sulfate polyacrylamide gels

Isocitrate was cleaved into succinate and glyoxylate by isocitrate lyase (ICL) in the glyoxylate cycle. We used lactate dehydrogenase as an ancillary enzyme to further metabolize the glyoxylate to glycolate in the presence of NADH. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and 2,6‐dichlorophenol‐indolphenol (DCPIP) were used in the coupling reactions for detecting ICL activity after electrophoresis on either native or sodium dodecyl sulfate (SDS) polyacrylamide gels. This fast and sensitive method can be used in the process of ICL enzyme purification and characterization.