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Performance of Lactococcus lactis as a starter culture in food production largely depends on the use of lyoprotectants during lyophilization. Gradient-freezing of bacterial cultures was conducted at 4°C, -20°C, and -80°C by storing the samples at each temperature for 2 h, successively. The entocytes extracted from the frozen cells were used as a lyoprotectant in the follow-up freeze-drying process of Lactococcus lactis subsp. lactis IL1403. The cell survival rate of gradient-freezing group increased 6.4-fold by bacterial plate count method. Furthermore, a proteomics and bioinformatics method was applied to elucidate the protein changes of Lactococcus lactis subsp. lactis IL1403 in response to gradient-freezing by gel-free proteomics using tandem mass tags (TMTs). The results showed that 121 stress-related proteins were significantly influenced by gradient-freezing. These proteins were involved in several metabolism pathways including ribosome metabolism, amino acid metabolism, quorum sensing, phosphotransferase system (PTS), pentose phosphate pathway, microbial metabolism in diverse environments, and nitrogen metabolism, etc. Some of these proteins especially the up-regulated proteins are potential lyoprotectants in vitro and they still need to be further investigated. [ABSTRACT FROM AUTHOR]

The archetypal membrane skeleton is that of the erythrocyte, consisting predominantly of spectrin, actin, ankyrin R and protein 4.1R. The presence in the Golgi of a membrane skeleton with a similar structure has been inferred, based on the identification of Golgi-associated spectrin and ankyrin. It has long been assumed that a Golgi-specific protein 4.1 must also exist, but it has not previously been found. We demonstrate here that a hitherto unknown form of protein 4.1, a 200 kDa 4.1B, is associated with the Golgi of Madin-Darby canine kidney (MDCK) and human bronchial epithelial (HBE) cells. This 4.1B variant behaves like a Golgi marker after treatment with Brefeldin A and during mitosis. Depletion of the protein in HBE cells by siRNA resulted in disruption of the Golgi structure and failure of Na+/K+-ATPase, ZO-1 and ZO-2 to migrate to the membrane. Thus, this newly identified Golgi-specific protein 4.1 appears to have an essential role in maintaining the structure of the Golgi and in assembly of a subset of membrane proteins. [ABSTRACT FROM AUTHOR]

The effects of tea polyphenols and ultraviolet radiation on the growth and antioxidant activity of mung bean sprouts were investigated, followed by the changes in corresponding antioxidant enzyme activities and metabolite composition. The growth of mung bean sprouts was slightly decreased by tea polyphenols and ultraviolet radiation, but the antioxidant activity was increased. The activities of peroxidase and catalase were decreased, and the contents of vitamin C, γ-aminobutyric acid and total phenols were significantly increased by tea polyphenol treatment. While the activities of peroxidase and catalase increased by ultraviolet radiation with concomitant increase in the contents of vitamin C and total phenols. The mung bean sprouts produced following imposition of stressors such as tea polyphenols and ultraviolet radiation from stress conditions could be used as a potential functional food. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndromes (MDSs) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Anemia is the defining cytopenia of MDS patients, yet the molecular mechanisms for dyserythropoiesis in MDSs remain to be fully defined. Recent studies have revealed that heterozygous loss-of-function mutation of DNA dioxygenase TET2 is 1 of the most common mutations in MDSs and that TET2 deficiency disturbs erythroid differentiation. However, mechanistic insights into the role of TET2 on disordered erythropoiesis are not fully defined. Here, we show that TET2 deficiency leads initially to stem cell factor (SCF)-dependent hyperproliferation and impaired differentiation of human colony-forming unit-erythroid (CFU-E) cells, which were reversed by a c-Kit inhibitor. We further show that this was due to increased phosphorylation of c-Kit accompanied by decreased expression of phosphatase SHP-1, a negative regulator of c-Kit. At later stages, TET2 deficiency led to an accumulation of a progenitor population, which expressed surface markers characteristic of normal CFU-E cells but were functionally different. In contrast to normal CFU-E cells that require only erythropoietin (EPO) for proliferation, these abnormal progenitors required SCF and EPO and exhibited impaired differentiation. We termed this population of progenitors "marker CFU-E" cells. We further show that AXL expression was increased in marker CFU-E cells and that the increased AXL expression led to increased activation of AKT and ERK. Moreover, the altered proliferation and differentiation of marker CFU-E cells were partially rescued by an AXL inhibitor. Our findings document an important role for TET2 in erythropoiesis and have uncovered previously unknown mechanisms by which deficiency of TET2 contributes to ineffective erythropoiesis. [ABSTRACT FROM AUTHOR]

The TET family of proteins plays important roles in a wide range of biological processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC). However, their function in erythropoiesis has remained unclear. We show here that TET2 and TET3, but not TET1 are expressed in human erythroid cells and we explore the role of these proteins in erythropoiesis. Knockdown experiments revealed that TET2 and TET3 have different functions. Suppression of TET3 expression in human CD34+ cells markedly impaired terminal erythroid differentiation, as reflected by increased apoptosis, generation of bi/multinucleated polychromatic/orthochromatic erythroblasts and impaired enucleation, though without effect on erythroid progenitors. In marked contrast, TET2 knockdown led to hyper-proliferation and impaired differentiation of erythroid progenitors. Surprisingly, knockdown of neither TET2 nor TET3 affected global levels of 5mC. Thus our findings have identified distinct roles for TET2 and TET3 in human erythropoiesis, and provide new insights into their role in regulating human erythroid differentiation at distinct stages of development. Moreover, as knockdown of TET2 recapitulates certain features of erythroid development defects characteristic of myelodysplastic syndromes (MDS), and TET2 gene mutation is one of the most common mutations in MDS, our findings may be relevant for improved understanding of dyserythropoiesis of MDS. [ABSTRACT FROM AUTHOR]

Protein 4.1G is a membrane skeletal protein that can serve as an adapter between transmembrane proteins and the underlying membrane skeleton. The function of 4.1G remains largely unexplored. Here, using 4.1G knockout mouse embryonic fibroblasts (MEFs) as a model system, we explored the function of 4.1G in motile cells. We show that the adhesion, spreading, and migration of 4.1G-/- MEF cells are impaired significantly. We further show that, although the total cellular expression of β1 integrin is unchanged, the surface expression of β1 integrin and its active form are decreased significantly in 4.1G-/- MEF cells. Moreover, the phosphorylation of focal adhesion kinase, a downstream component of the integrin-mediated signal transduction pathway, is suppressed in 4.1G-/- MEF cells. Co-immunoprecipitation experiments and in vitro binding assays showed that 4.1G binds directly to β1 integrin via its membrane-binding domain. These findings identified a novel role of 4.1G in cell adhesion, spreading, and migration in MEF cells by modulating the surface expression of β1 integrin and subsequent downstream signal transduction. [ABSTRACT FROM AUTHOR]

Background: The fermentation conditions of recombinant maltose-binding protein fused to neutrophil-activating protein (rMBP-NAP) of Helicobacter pylori were optimized from Escherichia coli TB1 with varying medium, inoculum age and size, time, inducer, pH and temperature in batch fermentation. Results: It was revealed that the optimal conditions for the production of rMBP-NAP in shake flask were as follows: M9 medium (with 3% yeast extract powder added), inoculum age of 19 h, inoculum size of 6%, initial pH of 6.6, temperature of 37°C, and 0.7 mmoL/L IPTG inducted 21 h in a 50 mL/250 mL shake flask. The recombinant protein yield was increased from 59 to 592 mg/L after optimization. Fermentation process conducted in a 10 L fermenter with similar conditions could get 30 g/L wet cell and 1.738 g/L soluble protein with the rMBP-NAP expression level of 11.9%. Conclusion: The results improve the expression level of rMBP-NAP, and it is expected that these optimized conditions can be well applied for large scale production of rMBP-NAP. [ABSTRACT FROM AUTHOR]