Leaf Mustard (Brassica juncea) Germplasm Resources Showed Diverse Characteristics in Agro-Morphological Traits and Glucosinolate Levels.

Leaf mustard, characterized by its purple/red/green leaves with a green/white midrib, is known for its thick, tender, and spicy leaves with a unique taste and flavor. There were only a few studies reported on leaf mustard for its morphological and biochemical traits from Korea. A total of 355 leaf mustard accessions stored at the GenBank of the National Agrobiodiversity Center were evaluated for 25 agro-morphological traits and seven intact glucosinolates (GSLs). The accessions showed a wide variation in terms of most of the traits. The quantitative agro-morphological traits varied from 16.0 (leaf length) to 48.7% (petiole width) of the coefficient of variation (CV). The highest variation was observed in glucoiberin (299.5%, CV), while the total GSL showed a CV of 66.1%. Sinigrin, followed by gluconapin and gluconasturtiin, was the most abundant GSL, accounting for as high as 75% of the total GSLs, while glucobrassicanapin and glucoiberin were the least abundant, contributing 0.7% and 0.1% on average, respectively. Sinigrin had a positive significant correlation with all GSLs but gluconasturtiin, while glucobarbarin and gluconasturtiin were highly positively correlated to each other, but least correlated with other GSLs. The leaf length was negatively correlated with sinigrin and glucoiberin. The width of the petiole showed a positive correlation with gluconapin, glucobrassicanapin, and glucobrassicin, while the length of the petiole had a negative correlation with sinigrin, glucobrassicanapin, glucoiberin, glucobrassicin, and the total GSLs. A higher width of the midrib was associated with higher contents of gluconapin, glucobrassicanapin, and glucobrassicin. A PCA analysis based on the agro-morphological traits showed that the first and second principal components accounted for 65.2% of the overall variability. Accessions that form a head tend to exhibit a longer leaf length, a larger plant weight, a thicker midrib, and higher widths of the midrib, petiole, and leaf. The GSLs showed inconsistent inter-and intra-leaf variation. Accessions that identified for various traits in their performance, such as, for example, Yeosu66 and IT259487 (highest total glucosinolates) and IT228984 (highest plant weight), would be promising lines for developing new varieties.

Supplementation with Vitis vinifera Jingzaojing Leaf and Shoot Extract Improves Exercise Endurance in Mice.

Switching myofibers from the fast-glycolytic type to the slow-oxidative type is associated with an alleviation of the symptoms associated with various cardiometabolic diseases. This study investigates the effect of Vitis vinifera Jingzaojing leaf and shoot extract (JLSE), which is rich in phenolic compounds, on the regulation of skeletal muscle fiber-type switching, as well as the associated underlying mechanism. Male C57BL/6N mice were supplemented orally with vehicle or JLSE (300 mg/kg) and subjected to treadmill exercise training. After four weeks, mice in the JLSE-supplemented group showed significantly improved exercise endurance and mitochondrial oxidative capacity. JLSE supplementation increased the expression of sirtuin 6 and decreased Sox6 expression, thereby elevating the number of mitochondria and encouraging fast-to-slow myofiber switching. The results of our experiments suggest that JLSE supplementation reprograms myofiber composition to favor the slow oxidative type, ultimately enhancing exercise endurance.

Glutamine deficiency shifts the asthmatic state toward neutrophilic airway inflammation.

BACKGROUND: The administration of L-glutamine (Gln) suppresses allergic airway inflammation via the rapid upregulation of MAPK phosphatase (MKP)-1, which functions as a negative regulator of inflammation by deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the role of endogenous Gln remains to be elucidated. Therefore, we investigated the mechanism by which endogenous Gln regulates MKP-1 induction and allergic airway inflammation in an ovalbumin-based murine asthma model. METHODS: We depleted endogenous Gln levels using L-γ-glutamyl-p-nitroanilide (GPNA), an inhibitor of the Gln transporter ASCT2 and glutamine synthetase small interfering siRNA. Lentivirus expressing MKP-1 was injected to achieve overexpression of MKP-1. Asthmatic phenotypes were assessed using our previously developed ovalbumin-based murine model, which is suitable for examining sequential asthmatic events, including neutrophil infiltration. Gln levels were analyzed using a Gln assay kit. RESULTS: GPNA or glutamine synthetase siRNA successfully depleted endogenous Gln levels. Importantly, homeostatic MKP-1 induction did not occur at all, which resulted in prolonged p38 MAPK and cytosolic phospholipase A2 (cPLA2 ) phosphorylation in Gln-deficient mice. Gln deficiency augmented all examined asthmatic reactions, but it exhibited a strong bias toward increasing the neutrophil count, which was not observed in MKP-1-overexpressing lungs. This neutrophilia was inhibited by a cPLA2 inhibitor and a leukotriene B4 inhibitor but not by dexamethasone. CONCLUSION: Gln deficiency leads to the impairment of MKP-1 induction and activation of p38 MAPK and cPLA2 , resulting in the augmentation of neutrophilic, more so than eosinophilic, airway inflammation.

Regulation of Alcohol and Acetaldehyde Metabolism by a Mixture of Lactobacillus and Bifidobacterium Species in Human.

Excessive alcohol consumption is one of the most significant causes of morbidity and mortality worldwide. Alcohol is oxidized to toxic and carcinogenic acetaldehyde by alcohol dehydrogenase (ADH) and further oxidized to a non-toxic acetate by aldehyde dehydrogenase (ALDH). There are two major ALDH isoforms, cytosolic and mitochondrial, encoded by ALDH1 and ALDH2 genes, respectively. The ALDH2 polymorphism is associated with flushing response to alcohol use. Emerging evidence shows that Lactobacillus and Bifidobacterium species encode alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) mediate alcohol and acetaldehyde metabolism, respectively. A randomized, double-blind, placebo-controlled crossover clinical trial was designed to study the effects of Lactobacillus and Bifidobacterium probiotic mixture in humans and assessed their effects on alcohol and acetaldehyde metabolism. Here, twenty-seven wild types (ALDH2*1/*1) and the same number of heterozygotes (ALDH2*2/*1) were recruited for the study. The enrolled participants were randomly divided into either the probiotic (Duolac ProAP4) or the placebo group. Each group received a probiotic or placebo capsule for 15 days with subsequent crossover. Primary outcomes were measurement of alcohol and acetaldehyde in the blood after the alcohol intake. Blood levels of alcohol and acetaldehyde were significantly downregulated by probiotic supplementation in subjects with ALDH2*2/*1 genotype, but not in those with ALDH2*1/*1 genotype. However, there were no marked improvements in hangover score parameters between test and placebo groups. No clinically significant changes were observed in safety parameters. These results suggest that Duolac ProAP4 has a potential to downregulate the alcohol and acetaldehyde concentrations, and their effects depend on the presence or absence of polymorphism on the ALDH2 gene.

Glucosinolate Content in Brassica Genetic Resources and Their Distribution Pattern within and between Inner, Middle, and Outer Leaves.

Glucosinolates (GSLs) are sulfur-containing secondary metabolites naturally occurring in Brassica species. The purpose of this study was to identify the GSLs, determine their content, and study their accumulation patterns within and between leaves of kimchi cabbage (Brassica rapa L.) cultivars. GSLs were analyzed using UPLC-MS/MS in negative electron-spray ionization (ESI-) and multiple reaction monitoring (MRM) mode. The total GSL content determined in this study ranged from 621.15 to 42434.21 µmolkg-1 DW. Aliphatic GSLs predominated, representing from 4.44% to 96.20% of the total GSL content among the entire samples. Glucobrassicanapin (GBN) contributed the greatest proportion while other GSLs such as glucoerucin (ERU) and glucotropaeolin (TRO) were found in relatively low concentrations. Principal component analysis (PCA) yielded three principal components (PCs) with eigenvalues ≥ 1, altogether representing 74.83% of the total variation across the entire dataset. Three kimchi cabbage (S/No. 20, 4, and 2), one leaf mustard (S/No. 26), and one turnip (S/No. 8) genetic resources were well distinguished from other samples. The GSL content varied significantly among the different positions (outer, middle, and inner) of the leaves and sections (top, middle, bottom, green/red, and white) within the leaves. In most of the samples, higher GSL content was observed in the proximal half and white sections and the middle layers of the leaves. GSLs are regarded as allelochemicals; hence, the data related to the patterns of GSLs within the leaf and between leaves at a different position could be useful to understand the defense mechanism of Brassica plants. The observed variability could be useful for breeders to develop Brassica cultivars with high GSL content or specific profiles of GSLs.

Rapid and Simultaneous Quantification of Five Quinolizidine Alkaloids in Lupinus angustifolius L. and Its Processed Foods by UPLC-MS/MS.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites of Lupinus plants. This study reports the simultaneous quantification of five alkaloids from Lupinus angustifolius L. and its processed foods by ultraperformance liquid chromatography with electrospray ionization mass spectrometric detection. After optimizing the extraction conditions, the analytical method was validated for the QAs in lupin beans and in three major processed foods through detection and quantification limits, linearity, precision, and accuracy. The detection and quantification limits of the QAs were 0.5-1.7 and 1.5-5.7 mg kg-1, respectively. The linearity was greater than 0.9992 and the precision was less than 3.1%. The recoveries of three different concentrations of each QA were 89.5-106.2%. For both raw lupin beans and processed foods, lupanine was the major QA, and 13-hydroxylupanine and angustifoline were detected in lower amounts. This method could be widely used for accurate risk assessment of QAs.

Houttuynia cordata Thunb Promotes Activation of HIF-1A-FOXO3 and MEF2A Pathways to Induce Apoptosis in Human HepG2 Hepatocellular Carcinoma Cells.

Houttuynia cordata Thunb ( H cordata), a medicinal plant, has anticancer activity, as it inhibits cell growth and induces cell apoptosis in cancer. However, the potential anti-cancer activity and mechanism of H cordata for human liver cancer cells is not well understood. Recently, we identified hypoxia-inducible factor (HIF)-1A, Forkhead box (FOX)O3, and MEF2A as proapoptotic factors induced by H cordata, suggesting that HIF-1A, FOXO3, and MEF2A contribute to the apoptosis of HepG2 hepatocellular carcinoma cells. FOXO3 transcription factors regulate target genes involved in apoptosis. H cordata significantly increased the mRNA and protein expression of HIF-1A and FOXO3 and stimulated MEF2A expression in addition to increased apoptosis in HepG2 cells within 24 hours. Therefore, we determined the potential role of FOXO3 on apoptosis and on H cordata-induced MEF2A in HepG2 cells. HIF-1A silencing by siRNA attenuated MEF2A and H cordata-mediated FOXO3 upregulation in HepG2 cells. Furthermore, H cordata-mediated MEF2A expression enhanced caspase-3 and caspase-7, which were abolished on silencing FOXO3 with siRNA. In addition, H cordata inhibited growth of human hepatocellular carcinoma xenografts in nude mice. Taken together, our results demonstrate that H cordata enhances HIF-1A/FOXO3 signaling, leading to MEF2A upregulation in HepG2 cells, and in parallel, it disturbs the expression of Bcl-2 family proteins (Bax, Bcl-2, and Bcl-xL), which results in apoptosis. Taken together, these findings demonstrate that H cordata promotes the activation of HIF-1A-FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells and is, therefore, a promising candidate for antitumor drug development.

Three degradation pathways of 1-octyl-3-methylimidazolium cation by activated sludge from wastewater treatment process.

The biodegradability and degradation pathways of 1-octyl-3-methylimidazolium cation [OMIM](+) by microbial community of wastewater treatment plant in Jeonju city, Korea were investigated. It was found that [OMIM](+) could be easily degraded by the microbial community. New degradation products and pathways of [OMIM](+) were identified, which are partially different from previous results (Green Chem. 2008, 10, 214-224). For the analysis of the degradation pathways and intermediates, the mass peaks observed in the range m/z of 50-300 were screened by using a tandem mass spectrometer (MS), and their fragmentation patterns were investigated by MS/MS. Surprisingly, we found three different degradation pathways of [OMIM](+), which were separated according to the initially oxidized position i.e. middle of the long alkyl chain, end of the long alkyl chain, and end of the short alkyl chain. The degradation pathways showed that the long and short alkyl chains of [OMIM](+) gradually degraded by repeating oxidation and carbon release. The results presented here shows that [OMIM](+) can be easily biodegraded through three different degradation pathways in wastewater treatment plants.

Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via nitric oxide production in human diploid fibroblasts.

Phenyl-2-pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near-senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose- and time-dependent manner and resulted in senescence-associated ß-galactosidase (SA-ß-gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence-associated proteins, such as phosphorylated ERK1/2, caveolin-1, p53, p16(ink4a), and p21(waf1), were elevated in PPKO-treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyloxy, and L-buthionine-(S,R)-sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L-NG-nitroarginine methyl ester and L-NG-monomethylarginine, PPKO-induced transient NO production and SA-ß-gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence-associated proteins.

Identification of a Polyketide Synthase Gene in the Synthesis of Phleichrome of the Phytopathogenic Fungus Cladosporium phlei.

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

ADP-ribose/TRPM2-mediated Ca2+ signaling is essential for cytolytic degranulation and antitumor activity of natural killer cells.

Natural killer (NK) cells are essential for immunosurveillance against transformed cells. Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable cation channel gated by ADP-ribose (ADPR). However, the role of TRPM2-mediated Ca(2+) signaling in the antitumor response of NK cells has not been explored. Here, we show that ADPR-mediated Ca(2+) signaling is important for cytolytic granule polarization and degranulation but not involved in target cell recognition by NK cells. The key steps of this pathway are: 1) the activation of intracellular CD38 by protein kinase A following the interaction of the NK cell with a tumor cell results in the production of ADPR, 2) ADPR targets TRPM2 channels on cytolytic granules, and 3) TRPM2-mediated Ca(2+) signaling induces cytolytic granule polarization and degranulation, resulting in antitumor activity. NK cells treated with 8-Br-ADPR, an ADPR antagonist, as well as NK cells from Cd38(-/-) mice showed reduced tumor-induced granule polarization, degranulation, granzyme B secretion, and cytotoxicity of NK cells. Furthermore, TRPM2-deficient NK cells showed an intrinsic defect in tumoricidal activity. These results highlight CD38, ADPR, and TRPM2 as key players in the specialized Ca(2+) signaling system involved in the antitumor activity of NK cells.

Seminal CD38 is a pivotal regulator for fetomaternal tolerance.

A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4(+) forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38(-/-) males was high compared with that in females mated with Cd38(+/+) males, and this was associated with a reduced proportion of Tregs within the CD4(+) T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.

Comparison of harmful gases produced during GreenLight High-Performance System laser prostatectomy and transurethral resection of the prostate.

OBJECTIVE: To compare the gases generated from GreenLight High-Performance System (HPS) laser prostatectomy with Urosol or normal saline solution and transurethral resection and vaporization of the prostate (TURVP) with Urosol. METHODS: A total of 36 smoke samples were collected from a continuous irrigation suction system attached to a Tenax absorber during transurethral surgery of the prostate. The gases were qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry equipped with a purge and trap sample injector. RESULTS: The gas produced during TURVP contained propylene, allene, isobutylene, 1,3-butadiene, vinyl acetylene, mercaptomethane, ethyl acetylene, diacetylene, 1-pentene, ethanol, piperylene, propenylacetylene, 1,4-pentadiene, cyclopentadiene, acrylnitrile, and butyrolacton. The types and amount of gas produced during HPS laser prostatectomy were fewer and smaller than during TURVP. However, 1,3-butadiene, a well-known human carcinogen, was also generated by HPS laser prostatectomy. HPS laser prostatectomy with saline produced a greater amount and number of gases than HPS laser prostatectomy with Urosol. CONCLUSION: The surgical smoke produced from TURVP and HPS laser prostatectomy contains potentially harmful chemical compounds, although HPS laser prostatectomy produced less surgical smoke than TURVP. Urosol produced fewer types and a smaller amount of gas than normal saline during HPS laser prostatectomy.

Comparative proteomic analysis for the insoluble fractions of colorectal cancer patients.

We used label-free quantitative proteomics with the insoluble fractions from colorectal cancer (CRC) patients to gain further insight into the utility of profiling altered protein expression as a potential biomarker for cancer. The insoluble fractions were prepared from paired tumor/normal biopsies from 13 patients diagnosed with CRC (stages I to IV). Fifty-six proteins identified in data pooled from the 13 cases were differentially expressed between the tumor and adjacent normal tissue. The connections between these proteins are involved in reciprocal networks related to tumorigenesis, cancer incidence based on genetic disorder, and skeletal and muscular disorders. To assess their potential utility as biomarkers, the relative expression levels of the proteins were validated using personal proteomics and a heat map to compare five individual CRC samples with five normal tissue samples. Further validation of a panel of proteins (KRT5, JUP, TUBB, and COL6A1) using western blotting confirmed the differential expression. These proteins gave specific network information for CRC, and yielded a panel of novel markers and potential targets for treatment. It is anticipated that the experimental approach described here will increase our understanding of the membrane environment in CRC, which may provide direction for making diagnoses and prognoses through molecular biomarker targeting.

Harmful gases including carcinogens produced during transurethral resection of the prostate and vaporization.

OBJECTIVE: To determine the chemical composition of surgical smoke produced during transurethral resection of the prostate (TURP) and vaporization. METHODS: A total of 12 smoke samples were collected from a continuous irrigation suction drainage system to a Tenax absorber at a 0.05L/min flow rate during TURP and vaporization. The gases were quantitatively and qualitatively analyzed by gas chromatography-mass spectrometry (GC-MS) equipped with a purge and trap sample injector. RESULTS: The main chemical constituents of surgical smoke produced during TURP and vaporization include propylene, allene, isobutylene, 1,3-butadiene, vinyl acetylene, mecaptomethane, ethyl acetylene, diacetylene, 1-pentene, EtOH, piperylene, propenylacetylene, 1,4-pentadiene, cyclopentadiene, acrylnitrile and butyrolactone. Three of the constituents are very toxic and carcinogenic (1,3-butadiene, vinyl acetylene and acrylonitrile). The amount (mean±standard deviation) of chemical components in the 45L of gas and room air mixture produced during TURP and vaporization were as follows: propylene, 0.80±0.52mg; isobutylene, 212.85±75.65mg; 1,3-butadiene, 0.93±0.34mg; ethyl acetylene, 0.09±0.05mg; 1-pentene, 6.75±1.62mg; 1,4-pentadiene, 0.06±0.02mg; and acrylonitrile, 1.62±1.19mg. CONCLUSIONS: Three of the toxic gases generated during TURP and vaporization are carcinogens (1,3-butadiene, vinyl acetylene and acrylonitrile). Therefore, higher quality filter masks, smoke evacuation devices and/or smoke filters should be developed for the safety of the operating room personnel and patients during TURP and vaporization.

Identification of metabolites involved in the biodegradation of the ionic liquid 1-butyl-3-methylpyridinium bromide by activated sludge microorganisms.

Ionic liquids (ILs) are low melting organic salts that potentially comprise wide application due to their fascinating properties and have emerged as promising "green" replacements for volatile organic solvents. Despite their nonmeasurable vapor pressure, some quantities of ILs will soon be present in effluent discharges since they do have significant solubility in water. Recently, the toxic effects of ILs toward aquatic communities have been intensively investigated, but little information is available concerning the biodegradable properties of these compounds. The objective of this study was to identify the metabolites generated during the biotransformation of 1-butyl-3-methylpyridinium by microorganisms in aerobic activated sludge. The obtained results revealed that the alkylpyridinium salt was metabolized through the sequential oxidization in different positions of the alkyl side chains. High-performance liquid chromatography and mass-spectrometry analyses demonstrated that this biodegradation led to the formation of 1-hydroxybutyl-3-methylpyridinium, 1-(2-hydroxybutal)-3-methylpyridinium, 1-(2-hydroxyethyl)-3-methylpyridinium, and methylpyridine. On the basis of these intermediate products, biodegradation pathways were also suggested. These findings provide the basic information that might be useful for assessing the factors related to the environmental fate and behavior of this commonly used pyridinium IL.

Proteomic analysis of recombinant Saccharomyces cerevisiae upon iron deficiency induced via human H-ferritin production.

In our previous study, the expression of active H-ferritins in Saccharomyces cerevisiae was found to reduce cell growth and reactive oxygen species (ROS) generation upon exposure to oxidative stress; such expression enhanced that of high-affinity iron transport genes (FET3 and FTR1). The results suggested that the recombinant cells expressing H-ferritins induced cytosolic iron depletion. The present study analyzes metabolic changes under these circumstances via proteomic methods. The YGH2 yeast strain expressing H-ferritin, the YGH2-KG (E62K and H65G) mutant strain, and the YGT control strain were used. Comparative proteomic analysis showed that the synthesis of 34 proteins was at least stimulated in YGH2, whereas the other 37 proteins were repressed. Among these, the 31 major protein spots were analyzed via nano-LC/MS/MS. The increased proteins included major heat-shock proteins and proteins related to endoplasmic reticulum-associated degradation (ERAD). On the other hand, the proteins involved with folate metabolism, purine and methionine biosynthesis, and translation were reduced. In addition, we analyzed the insoluble protein fractions and identified the fragments of Idh1p and Pgk1p, as well as several ribosomal assembly-related proteins. This suggests that intracellular iron depletion induces imperfect translation of proteins. Although the proteins identified above result from changes in iron metabolism (i.e., iron deficiency), definitive evidence for iron-related proteins remains insufficient. Nevertheless, this study is the first to present a molecular model for iron deficiency, and the results may provide valuable information on the regulatory network of iron metabolism.

Enhanced expression of high-affinity iron transporters via H-ferritin production in yeast.

Our heterologous expression system of the human ferritin H-chain gene (hfH) allowed us to characterize the cellular effects of ferritin in yeasts. The recombinant Saccharomyces cerevisiae (YGH2) evidenced impaired growth as compared to the control, which was correlated with ferritin expression and with the formation of core minerals. Growth was recovered via the administration of iron supplements. The modification of cellular iron metabolism, which involved the increased expression of high-affinity iron transport genes (FET3 and FTR1), was detected via Northern blot analysis. The findings may provide some evidence of cytosolic iron deficiency, as the genes were expressed transcriptionally under iron-deficient conditions. According to our results examining reactive oxygen species (ROS) generation via the fluorescence method, the ROS levels in YGH2 were decreased compared to the control. It suggests that the expression of active H-ferritins reduced the content of free iron in yeast. Therefore, present results may provide new insights into the regulatory network and pathways inherent to iron depletion conditions.

Induction of autoimmunity by immunization with hapten-modified hen egg lysozyme in hen egg lysozyme-transgenic mice.

To understand the mechanism of autoimmunity induction, hen egg lysozyme (HEL)-transgenic (Tg) C57BL/6 (B6) mice were immunized with HEL or phosphorylcholine-conjugated HEL (PC-HEL). Repeated immunization of HEL-Tg mice with native HEL failed to induce the antibody response against HEL. However, immunization with PC-HEL generated a significant anti-HEL antibody response. Immunization of the Tg mice with dominant (HEL(74-88)) or cryptic (HEL(47-61)) T-cell epitope peptide stimulated the corresponding T-cell response and similarly yielded the anti-HEL antibody response. Predominance of immunoglobulin G1 (IgG1) anti-HEL antibody response in the HEL-Tg mice and preferential IL-4 production by HEL-specific T cells suggested the dependency of the antibody response to the presence of T helper 2. HEL-Tg mice received HEL-primed B6 T cells, but not HEL-primed Tg T cells, were able to generate anti-HEL antibody response following PC-HEL immunization. The pattern and the level of epitope peptides generated by splenic antigen-presenting cells indicated that PC-HEL results in much more efficient processing as compared to HEL. These results strongly suggest that the enhancement of antigen processing by hapten (PC) conjugation to the antigen facilitates more efficient stimulation of T cells reactive to self antigen, HEL in HEL-Tg mice resulting in the production of anti-self HEL antibody.