• 한국육종학회지
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• 제41권3호
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• pp.205-212
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• 2009

Wheat (Triticum aestivum L.) grain texture is an important determinant of milling properties and end product use. Two linked genes, puroindoline a (PINA) and puroindoline b (PINB), control most of the genetic variation in wheat grain texture. Wheat seed proteins were examined to identify PINA and PINB gene using two pre-harvest sprouting wheat cultivars; Jinpum (resistant) and Keumgang (susceptible).Wheat seed proteins were separated by two-dimensional electrophoresis with IEF gels over pH ranges: pH 3-10. A total of 73 spots were digested with trypsin resulting peptide fragmentation were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS). Mass spectra were automatically processed and searched through NCBInr, SWISS-PORT and MSDB database with mono isotopic masses and complete gene sequence were found by UniProt database. Puroindoline a and puroindoline b that is responsible for grain texture related with baking performance and roughness. Two spots were found Pin b (16.7 kDa) and Pin a (16.3 kDa) in Jinpum compare to seven spots were identified Pin a (16.1 kDa, 16.3 kDa) and Pin b (16.7 kDa, 9.5 kDa and 14.4 kDa) in Keumgang. Some selected spots were identified puroindoline like grain softness protein (16.9 kDa, 17 kDa and 18.1 kDa) in Keumgang. Moreover, to gain a better inferring the identification of puroindoline related proteins using proteomics, we accomplished a complete gene sequence of PINA and PINB gene in pre-harvesting sprouting wheat seeds between resistant (Jinpum) and susceptible (Keumgang).

• Mass Spectrometry Letters
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• 제13권4호
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• pp.177-183
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• 2022

The monomer and dimer structures of the amyloid fragment Aβ(1-16) sequence formed in H₂O were investigated using electrospray ionization mass spectrometry (MS) and tandem MS (MS/MS). Aβ16 monomers and dimers were indicated by signals representing multiple proton adduct forms, [monomer+zH]ⁿ⁺ (=M^z+, z = charge state) and [dimer+zH]^z+ (=D^z+), in the MS spectrum. Fragment ions of monomers and dimers were observed using collision-induced dissociation MS/MS. Peptide bond dissociation was mostly observed in the D1-D7 and V11-K16 regions of the MS/MS spectra for the monomer (or dimer), regardless of the monomer (or dimer) charge state. Both covalent and non-covalent bond dissociation processes were indicated by the MS/MS results for the dimers. During the non-covalent bond dissociation process, the D³⁺ dimer complex was separated into two components: the M¹⁺ and M²⁺ subunits. During the covalent bond dissociation of the D³⁺ dimer complex, the b and y fragment ions attached to the monomer, (M+b_10-15)^z+ and (M+y_9-15)^z+, were thought to originate from the dissociation of the M²⁺ monomer component of the (M¹⁺+M²⁺) complex. Two different D³⁺ complex geometries exist; two distinguished interaction geometries resulting from interactions between the M¹⁺ monomer and two different regions of M²⁺ (the N-terminus and C-terminus) are proposed. Intricate fragmentation patterns were observed in the MS/MS spectrum of the D⁵⁺ complex. The complicated nature of the MS/MS spectrum is attributable to the coexistence of two D⁵⁺ configurations, (M¹⁺+M⁴⁺) and (M²⁺M³⁺), in the Aβ16 solution.

• Animal Bioscience
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• 제37권5호
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• pp.908-917
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• 2024

Objective: Although pork loins is not a tough meat, they need to develop meat products with a soft texture for the elderly. This study focused on the physicochemical properties and tenderness characteristics of pork loin injected with green kiwifruit juice (GRJ) and gold kiwifruit juice (GOJ) during various incubation times. In addition, the antioxidant activities of hydrolysate derived from the hydrolysis of pork loin by kiwifruit juice protease were evaluated. Methods: The pork loin was injected with 10% and 20% GRJ and GOJ, under various incubation times (0, 4, 8, and 24 h). Then, the physicochemical properties and tenderness of pork loins were measured. 2,2- diphenyl-1-picrylhydrazyl radical scavenging activity and reducing power were conducted to determine hydrolysate's antioxidant activities derived from pork loin's hydrolysis by kiwifruit juice protease. Results: GRJ had greater tenderizing ability than GOJ, even at the 10% addition. When kiwifruit juice was injected into pork loin, the tenderness increased with increasing incubation time. This was confirmed by the decrease in intensity of the myosin heavy chain (MHC) band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In particular, the MHC band decreased at 8 h for both 10% GRJ and 20% GOJ and at 4 h for 20% GRJ alone. The highest myofibril fragmentation index and peptide solubility were observed in pork loin treated with 20% GRJ compared to the other treatments during incubation. The 10% GRJ and 20% GOJ treatments showed similar levels of antioxidant activity of the protein hydrolysates in pork loin, and 20% GRJ showed the highest activity among the treatments. Conclusion: Kiwifruit juice had protease activity, and GRJ was more useful for tenderizing meat products than GOJ. Thus, GRJ at 10% could be a potential agent to tenderize and enrich the natural antioxidant activity through the proteolysis of pork loin.

• 대한한방종양학회지
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• 제6권1호
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.