Quantification of 4-methylimidazole in carbonated beverages by ultra-performance liquid chromatography-tandem mass spectrometry.

2- and 4-methylimidazoles (2-MI and 4-MI) are undesired byproducts produced during the manufacture of caramel color used to darken food products such as carbonated beverages. The Office of Environmental Health Hazard Assessment in California listed 4-MI as carcinogen in January 2011 with a proposed no significant risk level at 29 µg per person per day. Thus, a quantitative analytical measurement for 2-MI and 4-MI is desired for reliable risk assessments for exposure. An ultra-performance liquid chromatography (UPLC) coupled tandem mass spectrometric (MS/MS) method was developed for the quantification of 4-MI in beverage samples. Chromatographic separation of 2-MI and 4-MI were achieved by using a PFP reversed-phase column and a stepwise gradient of methanol and distilled water containing 0.1 % formic acid. Identification and quantification of 2-MI and 4-MI were performed using electrospray ionization-tandem mass monitoring the precursor to product ion transitions for 2-MI at m/z 83.1 → 42.2 and 4-MI at m/z 83.1 → 56.1 with melamine at m/z 127.1 → 85.1 as the internal standard. The performance of the method was evaluated against validation parameters such as specificity, carryover, linearity and calibration, correlation of determination (r(2)), detection limit, precision, accuracy, and recovery. Calibration curves at 10-400 ng/mL were constructed by plotting concentration versus peak-area ratio (analyte/internal standard) and fitting the data with a weighted 1/x. The accuracy of the assay ranged from 93.58 to 110.53 % for all analytes. Intra-assay precision for 2-MI and 4-MI were below 7.28 (relative standard deviation/RSD %) at QC samples. Here we present a new and improved method using UPLC-MS/MS to significantly simplify sample preparation and decrease chromatographic run time. This method allows accurate and reproducible quantification of 4-MI in carbonated beverages as low as sub ng/mL (ppb) levels.

E2F1-directed activation of Bcl-2 is correlated with lactoferrin-induced apoptosis in Jurkat leukemia T lymphocytes.

Lactoferrin (Lf) has been shown to control the proliferation of a variety of mammalian cells. Recently, we reported that human Lf induces apoptosis via a c-Jun N-terminal kinases (JNK)-associated Bcl-2 pathway that stimulates programmed cell death. In order to gain insight into the mechanism underlying Lf-triggered apoptotic features, we attempted to determine the mechanisms whereby the Lf-induced Bcl-2 family proteins exert their pro- or anti-apoptotic effects in Jurkat leukemia T lymphocytes. Treatment of the cells with high concentrations of Lf resulted in a significant reduction in in vitro growth and cell viability. As the levels of Lf increased, greater quantities of CDK6 and hyper-phosphorylated retinoblastoma protein were produced, resulting in the induction of E2F1-dependent apoptosis. Simultaneously, PARP and caspases were efficiently cleaved during Lf-induced apoptosis. The E2F1-induced apoptotic process occurred preferentially in p53-deficient Jurkat leukemia cells. Therefore, we attempted to determine whether E2F1-regulated Bcl-2 family proteins involved in the apoptotic process were relevant to Lf-induced apoptosis. We found that Lf increased the interaction of Bcl-2 with the pro-apoptotic protein Bad, whereas the total protein levels did not change significantly. Our results, collectively, suggest that Lf exploits the control mechanism of E2F1-regulated target genes or Bcl-2 family gene networks involved in the apoptotic process in Jurkat human leukemia T lymphocytes.

Iron-saturated lactoferrin stimulates cell cycle progression through PI3K/Akt pathway.

Iron binding lactoferrin (Lf) is involved in the control of cell cycle progression. However, the molecular basis underlying the effects of Lf on cell cycle control, as well as its target genes, remains incompletely understood. In this study, we have demonstrated that a relatively low level of ironsaturated Lf, Lf(Fe(3+)), can stimulate S phase cell cycle entry, and requires Akt activation in MCF-7 cells. Lf(Fe(3+)) immediately induced Akt phosphorylation at Ser473, which subsequently induced the phosphorylation of two G1-checkpoint Cdk inhibitors, p21(Cip/WAF1) and p27(kip1). The Lf(Fe(3+))-induced phosphorylation of Cdk inhibitors impaired their nuclear import behavior, thereby inducing cell cycle progression. However, the treatment of cells with a PI3K inhibitor, LY294002, almost completely blocked Lf(Fe(3+))-stimulated cell cycle progression. LY294002 treatment abrogated Lf(Fe(3+))-induced Akt activation, and prevented the cytoplasmic localization of p27(kip1). Higher levels of p21(Cip/WAF1) were also detected in the cytoplasmic sub-cellular compartment as a measure of cellular response to Lf(Fe(3+)). Consequently, the degree of phosphorylation of retinoblastoma protein was enhanced in response to Lf(Fe(3+)). Therefore, we conclude that Lf(Fe(3+)), as a potential antagonist of Cdk inhibitors, can facilitate the functions of E2F during progression to S phase via the Akt signaling pathway.

Requirement of the JNK-associated Bcl-2 pathway for human lactoferrin-induced apoptosis in the Jurkat leukemia T cell line.

The cell proliferation of p53-deficient Jurkat T cells is controlled after prolonged exposure to human lactoferrin (Lf). However, the molecular mechanism by which Lf influences these cellular responses remains unclear. In this study, we demonstrate that Lf-induced apoptosis in Jurkat T cells occurs in a dose- and time-dependent manner via the regulation of c-Jun N-terminal kinase (JNK) activity. Jurkat cells exposed to Lf for 1 day, especially at concentrations in excess of 500 microg/ml, showed typical apoptosis, as indicated by decreased cell viability and increased Annexin V binding. Our results also showed that Lf induced the activation of caspase 9 and caspase 3 activation, as demonstrated by our detection of cleaved caspases and PARP. Lf-induced apoptosis did not influence Bcl-2 expression via an ERK1/2 phosphorylation pathway, but was rather associated with the level of Bcl-2 phosphorylation. The treatment of cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor SB203580, revealed that the JNK-Bcl-2 signaling cascade is required for Lf-induced apoptosis. When JNK activation was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated Jurkat cells did not undergo cell death. These findings indicate that Lf functions as a biological mediator of apoptosis in the human leukemia Jurkat T-cell line, via the JNK-associated Bcl-2 signaling pathway.

Oxidative stress induces PKR-dependent apoptosis via IFN-gamma activation signaling in Jurkat T cells.

The dsRNA-dependent protein kinase, PKR, is a central component in antiviral defense. The biological importance of PKR is further remarked by its critical role in apoptosis induced by a variety of stresses. Here, we analyzed the implication of oxidative stress in the induction of PKR-dependent apoptosis in Jurkat cells. Our results revealed that reactive oxygen species (ROS) induced endogenous pkr gene expression at the transcriptional level by activating the interferon (IFN)-gamma gene. However, IFN-gamma siRNA expression abrogated the H(2)O(2)-mediated pkr induction. The radical scavenger N-acetyl-l-cysteine profoundly inhibited pkr induction via the reduction of IFN-gamma expression. The treatment of cells with the specific JAK-STAT inhibitor, AG490, reduced the PKR expression, and suppressed PKR-dependent cell death. Finally, siRNA-mediated depletion of IFN-gamma or pkr efficiently downregulated H(2)O(2)-mediated apoptotic cell death. These results indicated that oxidative stress induces PKR expression essentially via the IFN-gamma activation signal, and causes apoptosis in Jurkat T cells.

Preparation, swelling and electro-mechano-chemical behaviors of a gelatin-chitosan blend membrane.

Swelling, states of water, morphology, stability in the aqueous solution, and electro-mechano-chemical bending behaviors of the gelatin-chitosan blend system were studied in order to clarify the potential use of this blend system as an actuator. The gelatin-chitosan blend system was prepared in order to avoid dissolution of the pure chitosan in an aqueous medium and the rigidity and easy degradation of the pure gelatin in the swollen state. The blend systems showed improved material properties: the vacuum-dried blend sample at the G75/C25 (w/w, gelatin-chitosan) ratio showed ∼6 times swelling (in distilled water, at neutral pH and room temperature), ∼5 times stability (in distilled water), and ∼6 times bending (at 6 V/53 mm and in a 0.02 M NaCl aqueous solution) as compared to pure gelatin. These enhanced properties could be explained by the introduction of free -OH, -NH2, and -NHOCOCH3 groups of the amorphous chitosan in the blend and the network structure through the electrostatic interactions between the ammonium (-NH3+) ions of the chitosan and the carboxylate (-COO-) ions of the gelatin. The scanning electron microscopy (SEM) micrographs of the surfaces of the blend films showed homogeneous and smooth surfaces due to the good miscibility between gelatin and chitosan. However, a different morphology from the fractured surface was found between the pure gelatin and blend systems which showed condensed and foliaceous morphologies, respectively. The leafy morphology indicates a large and homogeneous pore structure, which would cause increased ion diffusion into the gel and might lead to increased bending.

Cutting edge: two distinct motifs within the Fas ligand tail regulate Fas ligand-mediated costimulation.

The cytoplasmic domain of Fas ligand is sufficient to costimulate CD8(+) T cells by driving Fas ligand recruitment into lipid rafts and association with select Src homology 3-containing proteins, activating PI3K and MAPK pathways, mediating nuclear translocation of the transcription factors NFAT and AP-1, and enhancing IFN-gamma production and Ag-specific CD8(+) T cell proliferation. We now show that Fas ligand molecules lacking amino acids 45-54 in the proline-rich region of the cytoplasmic domain fail to costimulate but serve as effective death inducers. Death induction and costimulation by Fas ligand are therefore clearly separable functions. Further, upon Fas ligand-mediated costimulation, casein kinase I phosphorylates Fas ligand, in which two conserved casein kinase I binding sites regulate NFAT activation and costimulation. These results help resolve how one molecule can serve as a double-edged immunomodulator by directing discrete biological consequences.

A distinct role of neutrophil lactoferrin in RelA/p65 phosphorylation on Ser536 by recruiting TNF receptor-associated factors to IkappaB kinase signaling complex.

The activation of NF-kappaB by neutrophil lactoferrin (Lf) is regulated via the IkappaB kinase (IKK) signaling cascade, resulting in the sequential phosphorylation and degradation of IkappaB. In this study, we observed that Lf protein augmented p65 phosphorylation at the Ser(536), but not the Ser(276) residue, and stimulated the translocation of p65 into the nucleus. Lf was also shown to enhance the association between p65 and CREB-binding protein/p300 in vivo. To elucidate the mechanism by which Lf triggers these signaling pathways, we attempted to delineate the roles of the upstream components of the IKK complex, using their dominant-negative mutants and IKKalpha(-/-) and IKKbeta(-/-) mouse embryonic cells. We demonstrated that both IKKalpha and IKKbeta as well as NF-kappaB-inducing kinase are indispensable for Lf-induced p65 phosphorylation. However, MAPK kinase kinase 1 is not essentially required for this activation. We also observed that Lf-induced p65 phosphorylation was either partially or completely abrogated as the result of treatment with the mutant forms of TNFR-associated factor (TRAF) 2, TRAF5, or TRAF6. Moreover, we demonstrated that Lf directly interacted with TRAF5. Expression of the dominant-negative mutant of TRAF5 or its small interfering RNA almost completely abrogated the Lf-induced p65 phosphorylation. These results suggest that signaling pathways, including TRAFs/NF-kappaB-inducing kinase/IKKs, may be involved in the regulation of Lf-induced p65 activation, thereby resulting in the activation of members of the NF-kappaB family.

Human lactoferrin controls the level of retinoblastoma protein and its activity.

Lactoferrin (Lf) has been implicated in the regulation of cell growth. However, the molecular mechanism underlying this effect remains to be elucidated. In this study, we show that Lf is involved in the cell cycle control system in a variety of cell lines, through retinoblastoma protein (Rb)--mediated growth arrest. We observed that Lf induces the expression of Rb, a signal mediator of cell cycle control, and that a majority of this Lf-induced Rb persists in a hypophosphorylated form. In addition, we determined that Lf specifically augments the level of a cyclin-dependent kinase inhibitor, p21, but not p27. Upon treatment with Lf, H1299 cells expressing defective p53 effected an augmentation of endogenous p21 levels, which may contribute to the accumulation of hypophosphorylated Rb. A substantial quantity of active Rb binds more efficiently to E2F1 in cells that express Lf and consequently blocks the expression of an E2F1-responsive gene, thereby suggesting that Lf plays a crucial role in the inhibition of tumor cell growth. Therefore, we conclude that the antiproliferative effects of Lf can likely be attributed to the elevated levels of hypophosphorylated Rb.

Phosphorylation of peptides derived from isolated soybean protein: effects on calcium binding, solubility and influx into Caco-2 cells.

Calcium homeostasis in the human body is maintained primarily via the absorption of calcium through the intestine. In order to maintain an efficient absorption of calcium with minimal calcium loss due to the formation of calcium phosphate precipitates in the small intestinal lumen, we developed a calcium-binding mediator using peptides derived from isolated soybean protein (ISP). ISP was modified via tryptic digestion and chemical phosphorylation using sodium trimetaphosphate, thereby generating soybean phosphopeptides (SPP), and this was followed by conducting a binding reaction with calcium chloride. We have established an optimized procedure and reaction conditions for maximal phosphorylation and calcium binding. Consequently, the phosphorylation of soybean peptides resulted in considerable improvement in their calcium binding activities. Next, we demonstrated that SPP was able to render calcium ions resistant to precipitate formation with inorganic phosphates, which suggested the enhancement of calcium bioavailability. Finally, we noted that the addition of calcium-bound SPP induced an increase in cytosolic calcium concentration in the intestinal Caco-2 cells, due to an influx of calcium. These findings provide a new basis by which we may assess the possibility that SPP, as a potent calcium carrier, can be utilized in the prevention of poor absorption of dietary calcium in animals.