Effects of high-pressure processing on the functional properties of pork batters containing Artemisia sphaerocephala krasch gum.

Here, the effect of high-pressure conditions (0.1-400 MPa) on the water-loss, texture, gel strength, color, dynamic rheological property, and water migration of pork batters containing 0.1% (W/W) Artemisia sphaerocephala krasch gum (PB-AG) is studied. Results indicated that the cooking yield, water-holding capacity, texture, gel strength, L* values, and G' values increased with the increase in pressure (0.1-300 MPa) (p < 0.05). Dynamic rheological results (G') revealed that the thermal gelling ability of the PB-AG gel gradually increased with pressure (0.1-300 MPa). The minimum of T22 content was observed and the proportion of immobilized water decreased at 300 MPa by low-filed nuclear magnetic resonance. However, excessive high-pressure processing treatments (400 MPa) resulted in lower gel strength, WHC, texture, and G'. The scanning electron microscopy results shown that a denser network structure with small cavities was observed at 300 MPa. Therefore, moderate pressure treatment (≤300 MPa) may improve gelation properties of PB-AG gel, while excessive pressure treatment (400 MPa) may weaken the gelation properties. PRACTICAL APPLICATION: High-pressure processing combining Artemisia sphaerocephala krasch gum could enhance the gelation properties of pork batters. To do so, establishing knowledge on gelation properties of pork batters with Artemisia sphaerocephala krasch gum at different pressure levels treatment would be of paramount importance, because this contributes furnishing engineering data pertinent to the technical progress for the processing of emulsion-type meat with high quality.

The effects of sodium chloride on proteins aggregation, conformation and gel properties of pork myofibrillar protein Running Head: Relationship aggregation, conformation and gel properties.

The objective of this study was to evaluate relationship with aggregation, secondary structures and gel properties of pork myofibrillar protein with different sodium chloride (1%, 2% and 3%). When the sodium chloride increased from 1 to 3%, the active sulfhydryl, surface hydrophobicity, hardness and cooking yield of myofibrillar protein were increased significantly (p < 0.05), the particle size, total sulfhydryl and Zeta potential were decreased significantly (p < 0.05), these meant the aggregations of pork myofibrillar protein were decreased. The changes of proteins aggregation induced the strongest intensity band of Amide I shifted up from 1660 cm-1 to 1661 cm-1, meanwhile, the ß-sheet structure content was increased significantly (p < 0.05) with the sodium chloride increased. From the above, the lower proteins aggregation and higher ß-sheet structure content could improve the water holding capacity and texture of pork myofibrillar protein gel.

Physicochemical Properties and Protein Denaturation of Pork Longissimus Dorsi Muscle Subjected to Six Microwave-Based Thawing Methods.

Physicochemical changes and protein denaturation were evaluated for pork longissimus dorsi muscle subjected to different thawing methods. Fresh pork longissimus dorsi muscle served as a control. Microwave (MT), microwave combined with ultrasonic (MUT), microwave combined with 35 °C water immersion (MIT), microwave combined with 4 °C refrigeration (MRT), microwave combined with air convection (MAT), and microwave combined with running water (MWT) were applied. All microwave-based methods excepted for MT avoided localized overheating. The changes in the water holding capacity (WHC), color, TBARS, and protein solubility were lowest with MAT. Differential scanning calorimetry (DSC) and dynamic rheological property measurements indicated, that the MAT samples changed only slightly and presented with complete peaks and high G' values compared with the other treatments. Thus, MAT may reduce protein denaturation associated with meat thawing. The results of this study indicated that MAT effectively shortens thawing time, preserves meat quality and uniformity, and could benefit the meat industry and those who consume its products.

[Establishment of hematological malignancy model in ex vivo cell culture].

The aim of this study was to develop an ex vivo cell culture system for establishing the hematological malignancy model. Mouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing 10% FCS without growth factors, or with one of the following growth factor combinations: (1) murine c-kit ligand (KL) plus human flt3 ligand (FL); (2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6 (3/T/G/H6); (3) KL/FL plus 3/T/G/H6. The flow cytometry was used to detect the ability of combinations of growth factors to complement the oncogene fusion protein to support self-renewal of the transfected cells. The results showed that the transfected cells could be amplified sustainably in the logarithmic growth way. The indicated combination of c-Kit ligand (KL) with flt-3 ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK, in addition to KL/FL, the self-renewal of p210 BCR-ABL transfected-marrow cells also required IL-3. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy. It is concluded that this study establishes a culture system ex vivo which provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.

[Long-term expansion of T cell progenitors by JAK3 gene transduced primitive hematopoietic cells in vitro].

OBJECTIVE: To explore whether activation of the JAK3-signaling pathway can stimulate long-term expansion of the earliest T cell progenitors from transduced primitive hematopoietic cells and evaluate their potential ability of committed differentiation. METHODS: A retrovirus vector (RV) containing JAK3 gene, two binding sites for chemical inducers of dimerization (AP20187), and green fluorescence protein (GFP), MGI-F(2)JAK3, was constructed. The RV vector MGI-F(2)JAK3 was then transduced into murine bone marrow hematopoietic cells. The transduced murine bone hematopoietic marrow cells were divided equally into four groups blank control group (No drug group), AP20187 group (added with AP20187 only), SCF group (added with stem cell factor only), and AP20187 + SCF group. Cytometry was used to detect the GFP marker and observe the survival of cells. The murine bone hematopoietic marrow cells expanded for 50 days were divided into 2 groups: one group was washed to remove the cytokines to observe their survival, and AP20187 + SCF was added into the culture fluid of other group. Cytometry and CELLQuest v3.1 software analysis were used to analyze the phenotype of the cells of AP20187 + SCF after 50 days' expansion. The C-kit(hi) CD44(+)CD25(-)TN cells after 50 days' expansion were further cultured under the condition of SCF + IL7 + IL3 for 5 days. The differentiation rate of Thy1.2 positive cells was observed by cytometry. Five Ly5.2 mice underwent radiation of the dose of 600 cGy, and then injected with the expanded cells into the thymus. Three weeks later the mice were killed and their thymus glands were taken out to prepare suspension of single cells to undergo cytometry to observe the proportions of GFP positive CD3 and CD4 mature T lymphocytes. RESULTS: One week later the cells of the No Drug group all died, the cells of the AP20187 group and SCF group died 2 - 4 weeks later, however, the cells of the AP20187 + SCF continued to grow and expanded up to 10(12)-fold after 50 days' culture. The cells of the AP20187 + SCF group with the cytokines washed died 2 more weeks later, and those with the cytokines washed and added with AP20187 + SCF continued to grow. The phenotype of the expanded proportion was identified as the earliest T cell progenitors expressing C-kit(hi) CD44(+) CD25(-)TN (triple negative). These progenitors could differentiate into Thy1.2 + T cells in the presence of SCF + IL-7 + IL-3 culture condition. 32% - 96% of the mice thymus cell were GFP positive, 5% +/- 0.8% of the thymus cells were GFP + CD(3) double positive, and 11.0% +/- 2.1% of the T lymphocytes were GFP + CD4(+) double positive. CONCLUSION: AP20187 combined with SCF mediating the activation of JAK3 signaling can dramatically expand the earliest T cell progenitors subpopulation, and acquires the capacity to induce the differentiation into T mature cells. This system may help understand the T cell biology and provide a fundamental basement for gene therapy to immunodeficiency disease in the future.

[Promotion of in vitro long term expansion of primitive multipotential hematopoietic cells by transduced JAK2 gene].

OBJECTIVE: To explore whether activation of the JAK2-signaling pathway can stimulate long-term expansion and regulation of hematopoietic stem cells/multipotential hematopoietic progenitor cells (HSC/MHPC), and evaluate their potential ability of committed differentiation. METHODS: A retrovirus vector (RV) which contains JAK2 gene and two binding sites for a chemical inducers of dimerization (AP20187) was constructed. JAK2 can be dimerized by adding AP20187. Female C57BL/6 mice were euthanized and marrow cells were harvested. The RV vector was then transduced into murine bone marrow cells. Following transduction, Transduced cells were divided equally into four groups as follow: (1) No drug group, (2) AP2018 alone group, (3) SCF + Flt3-Ligand group, (4) AP20187 + SCF + Flt3-Ligand group. The expanded cells were further analyzed by phenotype, committed differentiation, progenitor colony assay as well as colony forming unite-spleen. RESULTS: Only the group of AP20187 combined SCF and Flt3-Ligand have a significant sustained outgrowth. The cells reached at 10(19) fold on the day 80. The phenotype of expanded cells were checked by flow cytometry at various time points after two months in vitro culture and we repeated them in five separate experiments. Sca-1 was consistently expressed at the levels in 52% approximately 98%, while 56% approximately 69% of cells expressed c-kit, 40% approximately 85% expressed CD34, About 12% approximately 46% expressed B220, 6% approximately 17% expressed Gr1, 0% approximately 20% expressed TER119, 5% approximately 36% expressed CD41, 35% approximately 46% expressed CD11b and none expressed CD3. The expanded cells could differentiate into granulocytes, macrophages, erythocytes and megakaryocytes under different cytokines combination. They were also capable of forming BFU-E, CFU-GM, CFU-Mix and IL-7 responsive B-lymphoid colonies in methylcellulose colonies assay. Colonies forming unites-Spleen were obtained when the expanded cells injected into lethally irradiated mice. CONCLUSION: The JAK2-mediated transgenic hematopoietic cells could be expanded and regulated in long-term period, and they are capable of maintaining multipotential differentiation. This research is setting up a fundamental basement for cell therapy in the future.

[Long term regulated expansion and committed differentiation of JAK2 gene transfected hematopoietic stem/progenitor cells in vitro].

OBJECTIVE: To explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro. METHODS: A murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE + 86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE + 86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows: (1) control group; (2) AP20187 alone group; (3) SCF alone group and (4) AP20187 + SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD(34), Gr1, CD(11b), TER119, CD(41), B220 and CD(3) monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated. RESULTS: A significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187 + SCF group. Cells expanded up to 10(14)-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD(34), c-kit and Sca1, while were almost negative for Gr1, CD(11b), TER119, CD(41), B220 and CD(3). Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E, CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S. CONCLUSIONS: AP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.