Purification and characterization of two forms of urokinase

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.     

Alginate‐Based Emulsion Template Containing High Oil Loading Stabilized by Nonionic Surfactants

Oil‐in‐water (O/W) emulsion‐gel systems containing high oil payloads are of increasing interest for food applications because of the reduction in encapsulation cost, consumption frequency or volume of food products. This study shows a facile approach to prepare stable alginate‐based O/W emulsions at high oil loading using a mixture of nonionic surfactants (Tween 80 and Span 20) as a template to form gelled‐emulsions. The synergistic effects of alginate and surfactants on the O/W emulsion properties were evaluated in terms of oil droplet size and emulsion stability. At 2% (w/v) of alginate and 1% (w/v) of surfactants, the size distribution of oil droplets was narrow and monomodal, even at an oil loading of 70% (v/v). The emulsions formed were stable against phase separation. The oil droplet size could be further reduced to below 1 μm using a high‐shear homogenizer. The emulsions formed could be easily molded and gelled into solids of different shapes via ionic gelation. The findings of this study create possible avenues for applications in food industries.     

Effect of Gaseous Ozone on Papaya Anthracnose

Anthracnose caused by the fungus Colletotrichum gloeosporioides is considered as one of the most devastating postharvest disease of papaya. The aim of this study was to evaluate the effectiveness of gaseous ozone as a potential antifungal preservation technique to overcome anthracnose disease of papaya during cold storage. Different concentrations of ozone (0 (control), 0.04, 1.6, and 4 ppm) were applied for various exposure durations (48, 96, and 144 h). Radial mycelia growth and conidial germination were evaluated in vitro after fungal exposure to the different levels and durations of ozone. Significant inhibition in radial mycelia growth of C. gloeosporioides was observed (p?<?0.05) in all ozone treatments as compared to the control during 8 days of incubation at room temperature (25?±?3 °C). Ozone treatment of papaya fruit with 1.6-ppm ozone for 96 h delayed and simultaneously decreased the disease incidence to 40 % whereas disease severity was rated at 1.7, following 28 days of storage at 12?±?1 °C and 80 % relative humidity. The scanning electron microscopy showed that 4-ppm ozone caused disintegration of spore structure and did not affect the cuticular surface of fruit. Thus, ozone fumigation can reduce postharvest losses of papaya caused by anthracnose.     

Silver Nanoparticles Inhibit Sodium Uptake in Juvenile Rainbow Trout (Oncorhynchus mykiss)

The silver ion (Ag(+)) is well documented to be a potent inhibitor of sodium (Na(+)) transport in fish. However, it has not been determined whether silver nanoparticles (Ag NPs) elicit this same effect and, if so, if the NP itself and/or the dissociation of ionic Ag(+) causes this effect. Citrate-capped Ag NPs were dialyzed in water to determine the dissolution rate of ionic Ag(+) from the NPs and the maximum concentration of free Ag(+) released from the NPs was used as a paired Ag(+) control to distinguish NP effects from ionic metal effects. The maximum concentration of ionic Ag(+) released from these NPs over 48 h was 0.02 μg l(-1). Juvenile rainbow trout were exposed to 1.0 mg l(-1) citrate-capped Ag NPs and dialyzed citrate-capped Ag NPs or 10 μg l(-1) and 0.02 μg l(-1) ionic Ag(+) (as AgNO(3)) as controls. Both nondialyzed and dialyzed Ag NPs and 10 μg l(-1) ionic Ag(+) significantly inhibited unidirectional Na(+) influx by over 50% but had no effect on unidirectional Na(+) efflux. Na(+),K(+)-ATPase was significantly inhibited by the Ag NPs with no discernible effect on carbonic anhydrase activity. This study is the first to show that sodium regulation is disrupted by the presence of citrate-capped Ag NPs, and the results suggest that there are nanospecific effects.     

Polyphenolic composition and in vitro antioxidant activities of native‐ and tannase‐treated green tea extracts

The antioxidant activities of native‐ and tannase‐treated green tea extracts along with their major polyphenol components were investigated. The polyphenolic content and composition of the tea before and after tannase treatment were determined by liquid chromatography coupled with mass spectrometry (LC‐MS). Approximately 99% of the (?)‐epigallocatechin gallate (EGCG) and (?)‐epicatechin gallate (ECG) in green tea extract were converted by tannase to (?)‐epigallocatechin (EGC) and (?)‐epicatechin (EC), respectively, after 30 min. Biotransformed green tea exhibited a significantly higher DPPH˙ radical scavenging activities than native green tea (EC₅₀ value of 0.024 ± 0.001 and 0.044 ± 0.001 mg mL^?1, respectively). Kinetic parameters such as scavenging rate and stoichiometry were calculated. The rate of DPPH˙ radical scavenging activities for tannase‐treated green tea extract was shown to be higher than native green tea extract.     

Antioxidant activity and profiles of common fruits in Singapore

Thirty-eight types of fruits commonly consumed in Singapore were systematically analysed for their hydrophilic oxygen radical absorbance capacity (H-ORAC), total phenolic content (TPC), ascorbic acid (AA) and various lipophilic antioxidants. Antioxidant composition and concentration varied widely across different fruits. Many of the tropical fruits tested were high in antioxidants. Amongst all fruits tested, sapodilla (Manilkara zapota) had the highest H-ORAC and TPC whilst guava had the highest AA per gram fresh weight. Papaya, red watermelon and cantaloupe had the highest β-cryptoxanthin, lycopene and β-carotene per gram fresh weight, respectively. On the other hand, durian and mangosteen were high in tocopherols and tocotrienols, respectively. Based on consumption data, Chinese Singaporeans appear to have a higher intake of carotenoids and tocopherols rich fruits compared to the US population. As fruits are a rich source of diverse antioxidants, efforts to promote consumption of a variety of fruits should be continued for public health benefits.     

Survival of SA11 rotavirus in fresh fruit juices of pineapple, papaya, and honeydew melon

Survival of rotavirus in fresh fruit juices of papaya (Caraca papaya L.), honeydew melon (Cucumis melo L.), and pineapple (Ananas comosus [L.] Merr.) was studied. Clarified juices were prepared from pulps of ripe fruits and sterilized by ultrafiltration. One milliliter of juice from each fruit was inoculated with 20 microl of 1 x 10(6) PFU of SA11 rotavirus and sampled immediately (0-h exposure) and 1 and 3 h later at 28 degrees C. Mean viral titers in juices of papaya (pH 5.1) and honeydew melon (pH 6.3) at 1 and 3 h were not significantly different from titers at 0-h exposure. Mean viral titers in juices from pineapples with ripening color indices of 3 (pH 3.6) and 6 (pH 3.7) at 1-h exposure (color index 3: 4.0 +/- 1.7 x 10(4); color index 6: 2.3 +/- 0.3 x 10(5)) and 3-h exposure (color index 3: 1.1 +/- 0.4 x 10(4); color index 6:1.3 +/- 0.6 x 10(5)) were significantly lower than titers at 0-h exposure (color index 3: 5.7 +/- 2.9 x 10(5); color index 6: 7.4 +/- 1.3 x 10(5)). Virus titers in pineapple juices of color index 3 were significantly lower than titers of the virus in juices of index 6. In cell culture medium (pH 7.4), SA11 titer remained stable over 3 h at 28 degrees C. However, at pH 3.6, the virus titer was reduced to a level not significantly different from that of the virus in pineapple juice of color index 6 (pH 3.7). In conclusion, papaya and honeydew melon juices, in contrast to pineapple juice, have the potential to transmit rotavirus. Inactivation of SA11 virus in pineapple juice can be possibly attributed to low pH and constituent(s) in the juice.     

THZ531 Induces a State of BRCAness in Multiple Myeloma Cells: Synthetic Lethality with Combination Treatment of THZ 531 with DNA Repair Inhibitors

Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12–THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.     

Reduced migration from flexible poly(vinyl chloride) of a plasticizer containing beta-cyclodextrin derivative

The migration of endocrine-disrupting di-(2-ethylhexyl) phthalate (DEHP) poses a serious threat to public health and the environment. In this study, we successfully prepared a plasticizerwith reduced DEHP migration by directly incorporating 2,3,6-per-O-benzoyl-beta-cyclodextrin (Bz-beta-CD) into DEHP. Bz-beta-CD was prepared by esterification between the hydroxyl groups of beta-CD and benzoyl chloride. The presence of this cyclodextrin is expected to facilitate formation of stable complexes through pi-pi association with DEHP molecules. The flexible PVC was prepared with a gelation-fusion process that uses the prepared migration-resistant plasticizer, and its properties (flexibility, thermal stability, and clarity) were evaluated by carrying out DSC and tensile testing, TGA, and haze testing, respectively. No significant changes in the physical properties of the flexible PVC were observed when Bz-beta-CD was added. DEHP migration tests were carried out for the flexible PVC according to the ISO 3826:1993(E) test method, and the quantity of migrated DEHP was then determined with UV-vis spectroscopy. It was found that the addition of Bz-beta-CD decreases the levels of DEHP migration from the flexible PVC samples by almost 40%. We investigated the molecular interaction between Bz-beta-CD and DEHP using molecular mechanics simulations, and we conclude that this reduction in DEHP migration is due to the formation of stabilized pi-pi attractive association and inclusion complexes of Bz-beta-CD and DEHP in flexible PVC.     

Incorporation and metabolic conversion of erucic acid in various tissues of the rat in short term experiments

Rats were intravenously injected with a mixture of free (14-¹⁴C) erucic acid (22∶1) and (9–10³H) oleic acid (18∶1). After 2, 4, 8, 16, and 30 min, radioactivity was examined in blood, liver, heart, kidneys, and spleen. Free (¹⁴C) 22∶1 disappeared from the blood more rapidly than free (¹⁴C) 18∶1 between 0 and 8 min. Incorporation of label into triglycerides only appeared after 16 min and at 30 min they represented 4% of the injected radioactivity. In this fraction, 63% of¹⁴C radioactivity was present as 18∶1 and not as the original 22∶1, while almost all³H radioactivity was recovered as unchanged 18∶1. At all times studied, the majority of radioactivity was found in the liver, primarily as triglycerides (60% of radioactivity in total lipids) and as phospholipids (20–30%).¹⁴C was present in nearly the same proportion as³H (13% of injected radioactivity after only 2 min, 11% at 30 min).¹⁴C radioactivity was contained in 18∶1 in higher proportion than 22∶1 (45% in triglycerides, 65% in phospholipids). Since labeled triglycerides of blood, rich in (¹⁴C) 18∶1, mainly originate from the liver triglycerides, it appears that 18∶1 is the major form of utilization of 22∶1 in the tissues after its conversion in liver. In the other organs tested, radioactivity was found 10–15 times lower than in liver. In the heart,¹⁴C was 3 to 4 times higher than³H. More than 80% was recovered as 22∶1 in triglycerides. In spleen and kidneys, the¹⁴C:³H ratio was particularly high in free fatty acids and monoglycerides. In kidneys, 60% of¹⁴C was present as nervonic acid (24∶1) in monoglycerides and 40% in phospholipids, suggesting that the mononervonin formed was used for phospholipid biosynthesis. A preliminary report of this work was presented at the 10th International Congress of Nutrition, Kyoto, Japan, August 1975.