Affinity Purification of Bovine Lactoferrin and Bovine Transferrin from Using Immobilized Gangliosides

Bovine lactoferrin (BLF) and bovine transferrin (BTF) are major-iron transport and regulation proteins found in bovine whey. BLF and BTF must interact with the eukaryotic cell surface to mediate their biological function of iron delivery and cellular functions of inflammatory and immunological modulation. As common components of the eukaryotic cell surface, gangliosides were used for affinity purification of BLF and BTF. Bovine gangliosides were isolated from fresh buttermilk and covalently immobilized onto controlled-pore glass beads (66 μg/g beads). After the matrix was loaded with whey protein (WPI or WPC), lactoferrin was eluted with 1 M NaCl and lll identified by N-terminal protein sequencing. Pretreated whey isolate (1 % wt/vol) showed the highest lactoferrin purity with 40% among protein sources, and whey protein isolate (10% wt/vol) showed the highest recovery with 105%. Bovine transferrin was eluted with sodium phosphate buffers at pH 7 after the immobilized matrix was loaded with a 2% (wt/vol) whey solution. The ganglioside column resulted in a 74.2% recovery of BTF from whey, and the BTF was enriched to 61% purity after Mono-Q chromatography. Bovine transferrin was identified by SDS-PAGE analysis, Western analysis, and isoelectrofocusing. In conclusion, immobilized gangliosides can be used to purify BLF and BTF from bovine whey

Affinity Purification and Characterization of \u3cem\u3eE. coli\u3c/em\u3e Molecular Chaperones

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well documented affinity for proteins lacking tertiary structure. Heat-induced Escherichia coli BL21 cell lysate (10 mg protein) was applied to immobilized ɑ-casein (45 mg/g beads) or β-casein (30 mg/g beads) column. After removing a majority of nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. Western analysis identified five Escherichia coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The β-Casein column showed a higher binding capacity than the ɑ-casein column since β-casein urea eluates contained 3.18 mg total protein (or 58% chaperone) compared to a-casein urea eluates with 2.68 mg total protein (or 32% chaperone). For strain comparison, Escherichia coli NM522 eluates showed more unidentified proteins in cold water eluates from both affinity columns. Chaperones were induced from BL21 strain with three treatments: heat shock at 39°C, heat shock at 42°C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized β-casein (30 mg/g beads) column. The molecular chaperones were eluted with cold water or 1 mM Mg-ATP after washing with 1 M NaCl. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. The treatment at 42°C was the most efficient for chaperone induction with highest chaperone yield of 1.0 mg among samples. Refolding denatured carbonic anhydrase B enzyme in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured enzyme and a 68% recovery of chemically denatured enzyme. It was concluded that the novel casein affinity chromatography is a rapid and efficient method for purification of chaperone. The affinity purified chaperones were effective in vitro folding aids

Antioxidant and whitening activities of five unripe pear cultivars

The present study was the first evaluation of the arbutin content, antioxidant activity, and whitening function of the unripe pears of five major Korean pear cultivars. Unripe pears were investigated 30 days after florescence for possible utilization as a whitening ingredient, instead of being thrown away for thinning out. Among the five cultivars tested, Gamcheonbae and Manpungbae had significantly higher total phenolics and arbutin contents, while Niitaka had lower values of both total phenolics and arbutin. For whitening activity related to tyrosinase and cellular melanin formation, Manpungbae also showed the strongest tyrosinase inhibition (4.9 %), and achieved 74% reduction of the cellular melanin compared to non-treated cells. These results indicate that unripe pears, especially the Manpungbae cultivar, could be useful for application as a possible natural whitening additive with high arbutin content and excellent whitening activity

Physiochemical, nutritional and functional characterization of 10 different pear cultivars (<i>Pyrus</i> spp.)

This study was performed to compare the physiochemical properties and nutritional components including sugars, amino acids, and minerals of 10 common pear cultivars cultivated in Korea (four Pyrus spp.). Furthermore, the pear cultivars were characterized for functional properties with respect to phenolic compounds by HPLC/DAD analysis and antioxidant activities using DPPH and ABTS assays. Among the 10 pear cultivars that were tested, Niitaka and Hanareum pears show the best physiochemical properties such as higher sugar/acid ratio and proper firmness. They also showed relatively enriched soluble sugar (12.6 ~ 13.0 g/100g FW), amino acid (4.5 ~ 7.3 g/100 g DW) or mineral contents with high K/Na ratio. For functional properties, Niitaka and Hanareum pears have significantly higher contents of total phenolics (240 mg/100 g DW), arbutin (103 ~ 124 mg/100 g DW), and chlorogenic acid (11 mg/100 g DW) as well as strong antioxidant activities (49 % or 86 %) among cultivars. These results indicate that Niitaka, and Hanareum cultivars, could be best for consumption or favorable processing due to excellent product quality and high concentrations of nutritional and functional compounds

Nutritional and neuroprotective characterization of 'Tadanishiki' yuzu according to harvesting period or extraction condition

The present study investigated the phenolic profile, antioxidant activity, and neuroprotective properties of ‘Tadanishiki’ yuzu (Citrus junos, a seedless variety of yuzu) according to harvesting period and extraction condition. High-performance liquid chromatography (HPLC) was used to identify the functional components. To evaluate the neuroprotective properties, scopolamine was used to induce cholinergic dysfunction in human neuroblastoma SH-SY5Y cells pretreated with yuzu extracts. Among the harvesting periods, September provided the optimum fruit weight of yuzu and relatively high amounts of total phenolics (3.67 mg/g DW), flavonoids (10.13 mg/g DW), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (29.10 μg Vit. C eq.). Of the functional compounds, hesperidin (13.57 mg/100 g DW) and naringin (5.84 mg/100 g DW) were the highest in 5% (w/v) yuzu extracted with 80% ethanol and this extract showed the highest DPPH (289.2 μg Vit. C eq.) scavenging activity. This same extract showed the highest cell viability and lowest cortisol or acetylcholinesterase content in scopolamine-treated SH-SY5Y cells. These results indicate that ‘Tadanishiki’ yuzu harvested in September should be extracted at 5% (w/v) yuzu with 80% EtOH, and this extract might be useful for application as a natural functional additive

Input of terrestrial organic matter linked to deglaciation increased mercury transport to the Svalbard fjords

Deglaciation has accelerated the transport of minerals as well as modern and ancient organic matter from land to fjord sediments in Spitsbergen, Svalbard, in the European Arctic Ocean. Consequently, such sediments may contain significant levels of total mercury (THg) bound to terrestrial organic matter. The present study compared THg contents in surface sediments from three fjord settings in Spitsbergen: Hornsund in the southern Spitsbergen, which has high annual volume of loss glacier and receives sediment from multiple tidewater glaciers, Dicksonfjorden in the central Spitsbergen, which receives sediment from glacifluvial rivers, and Wijdefjorden in the northern Spitsbergen, which receive sediments from a mixture of tidewater glaciers and glacifluvial rivers. Our results showed that the THg (52 +/- 15 ng g(-1)) bound to organic matter (OM) was the highest in the Hornsund surface sediments, where the glacier loss (0.44 km(3) yr(-1)) and organic carbon accumulation rates (9.3 similar to 49.4 g m(-2) yr(-1)) were elevated compared to other fjords. Furthermore, the delta C-13 (-27 similar to -24 parts per thousand) and delta S-34 values (-10 similar to 15 parts per thousand) of OM indicated that most of OM were originated from terrestrial sources. Thus, the temperature-driven glacial melting could release more OM originating from the meltwater or terrestrial materials, which are available for THg binding in the European Arctic fjord ecosystems.11Ysciescopu

Generation of a single-cycle pulse using a two-stage compressor and its temporal characterization using a tunnelling ionization method

A single-cycle laser pulse was generated using a two-stage compressor and characterized using a pulse characterization technique based on tunnelling ionization. A 25-fs, 800-nm laser pulse was compressed to 5.5 fs using a gas-filled hollow-core fibre and a set of chirped mirrors. The laser pulse was further compressed, down to the single-cycle limit by propagation through multiple fused-silica plates and another set of chirped mirrors. The two-stage compressor mitigates the development of higher-order dispersion during spectral broadening. Thus, a single-cycle pulse was generated by compensating the second-order dispersion using chirped mirrors. The duration of the single-cycle pulse was 2.5 fs, while its transform-limited duration was 2.2 fs. A continuum extreme ultraviolet spectrum was obtained through high-harmonic generation without applying any temporal gating technique. The continuum spectrum was shown to have a strong dependence on the carrier-envelope phase of the laser pulse, confirming the generation of a single-cycle pulse. © 2019, The Author(s

Genetic Mechanisms in Aspirin-Exacerbated Respiratory Disease

Aspirin-exacerbated respiratory disease (AERD) refers to the development of bronchoconstriction in asthmatics following the exposure to aspirin or other nonsteroidal anti-inflammatory drugs. The key pathogenic mechanisms associated with AERD are the overproduction of cysteinyl leukotrienes (CysLTs) and increased CysLTR1 expression in the airway mucosa and decreased lipoxin and PGE2 synthesis. Genetic studies have suggested a role for variability of genes in disease susceptibility and the response to medication. Potential genetic biomarkers contributing to the AERD phenotype include HLA-DPB1, LTC4S, ALOX5, CYSLT, PGE2, TBXA2R, TBX21, MS4A2, IL10, ACE, IL13, KIF3A, SLC22A2, CEP68, PTGER, and CRTH2 and a four-locus SNP set composed of B2ADR, CCR3, CysLTR1, and FCER1B. Future areas of investigation need to focus on comprehensive approaches to identifying biomarkers for early diagnosis