Cytochrome P450 2J2, a new key enzyme in cyclophosphamide bioactivation and a potential biomarker for hematological malignancies.

The role of cytochrome P450 2J2 (CYP2J2) in cyclophosphamide (Cy) bioactivation was investigated in patients, cells and microsomes. Gene expression analysis showed that CYP2J2 mRNA expression was significantly (P<0.01) higher in 20 patients with hematological malignancies compared with healthy controls. CYP2J2 expression showed significant upregulation (P<0.05) during Cy treatment before stem cell transplantation. Cy bioactivation was significantly correlated to CYP2J2 expression. Studies in HL-60 cells expressing CYP2J2 showed reduced cell viability when incubated with Cy (half maximal inhibitory concentration=3.6 mM). Inhibition of CYP2J2 using telmisartan reduced Cy bioactivation by 50% and improved cell survival. Cy incubated with recombinant CYP2J2 microsomes has resulted in apparent Km and Vmax values of 3.7-6.6 mM and 2.9-10.3 pmol/(min·pmol) CYP, respectively. This is the first study demonstrating that CYP2J2 is equally important to CYP2B6 in Cy metabolism. The heart, intestine and urinary bladder express high levels of CYP2J2; local Cy bioactivation may explain Cy-treatment-related toxicities in these organs.

Inhibition of Ephrin B3-mediated survival signaling contributes to increased cell death response of non-small cell lung carcinoma cells after combined treatment with ionizing radiation and PKC 412.

Radiation therapy is frequently used to treat non-small cell lung cancers (NSCLCs). We have previously shown that a combination of ionizing radiation (IR) and the staurosporine analog PKC 412, but not Ro 31-8220, increases cell death in NSCLC cells. To identify genes involved in the enhancement of cell death, a total gene profiling in response to co-administration of (i) PKC 412 with IR, or (ii) Ro 31-8220 with IR was implemented. These combined treatments caused upregulation of 140 and 179 genes and downregulation of 253 and 425 genes, respectively. Certain genes were selected and verified by real-time quantitative PCR and, of these genes, robust suppression of Ephrin B3 expression was suggested as a possible cell death-inducing mechanism of combined treatment with IR and PKC 412. Indeed, silencing of Ephrin B3 using siRNA in NSCLC cells resulted in a major alteration of their morphology with an elongated phenotype, decreased proliferation and increased cell death signaling. Moreover, silencing of Ephrin B3 in combination with IR caused a decrease in IR-mediated G(2)-arrest, induced cellular senescence, inhibited MAPK ERK and p38 phosphorylation, and caused an upregulation of p27(kip1) expression. Finally, silencing of Ephrin B3 in combination with IR sensitized U-1810 cells to IR-induced apoptosis. In conclusion, we identify and describe Ephrin B3 as a putative signaling molecule involved in the response of NSCLC cells to combined treatment with PKC 412 and ionizing radiation.

Early-phase GVHD gene expression profile in target versus non-target tissues: kidney, a possible target?

GVHD is a major complication after allo-SCT. In GVHD, some tissues like liver, intestine and skin are infiltrated by donor T cells while others like muscle are not. The mechanism underlying targeted tropism of donor T cells is not fully understood. In the present study, we aim to explore differences in gene expression profile among target versus non-target tissues in a mouse model of GVHD based on chemotherapy conditioning. Expression levels of JAK-signal transducers and activators of transcription (STAT), CXCL1, ICAM1 and STAT3 were increased in the liver and remained unchanged (or decreased) in the muscle and kidney after conditioning. At the start of GVHD the expression levels of CXCL9, ITGb2, SAA3, MARCO, TLR and VCAM1 were significantly higher in the liver or kidney compared with the muscle of GVHD animals. Moreover, biological processes of inflammatory reactions, leukocyte migration, response to bacterium and chemotaxis followed the same pattern. Our data show that both chemotherapy and allogenicity exclusively induce expression of inflammatory genes in target tissues. Moreover, gene expression profile and histopathological findings in the kidney are similar to those observed in the liver of GVHD mice.

miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK, apoptosis and senescence.

BACKGROUND: Radio- and chemotherapy (RT/CT) resistance hampers success in combating small and non-small cell lung cancers (SCLC/NSCLC). The underlying molecular mechanisms of RT/CT resistance of LCs are multifactorial and have been understood in part hitherto. miRNAs, key regulators of mRNAs, are well-recognised oncomirs; however, their role in regulating RT response remains poorly understood. METHODS: Six human NSCLC and five SCLC cell lines with different SF2 values were investigated. Using microarray we examined whether expression of miRNAs is linked to the RT resistance of NSCLCs or SCLCs. Obtained data were validated by quantitative real-time PCR. Apoptosis and senescence were analysed using siRNA transfection, western blot and flow cytometry. RESULTS: miRNA-21, miRNA-1827, miRNA-214, miRNA-339-5p, miRNA-625, miRNA-768-3p, miRNA-523-3p, miRNA-1227, miRNA-324-5p, miRNA-423-3p, miRNA-1301 and miRNA-1249 are differentially expressed in LC cells. miRNA-214 is upregulated in RT-resistant NSCLC cells relative to radiosensitive counterparts. Considering miRNA-214 as a putative regulator of RT resistance, we demonstrate that knockdown of miRNA-214 in radioresistant NSCLCs sensitised them to RT by stimulation of senescence. Consistently, overexpression of miRNA-214 in radiosensitive NSCLCs protected against RT-induced apoptosis. Protection was mediated by p38MAPK, as downregulation of this kinase could reverse the miRNA-214 overexpression-induced resistance of NSCLC cells. CONCLUSION: miRNA profiling of LC revealed putative RT resistance signalling circuits, which might help in sensitisation of LC to RT.

Nicotine improves ethanol-induced impairment of memory: possible involvement of nitric oxide in the dorsal hippocampus of mice.

In the present study, the possible involvement of nitric oxide (NO) systems in the dorsal hippocampus in nicotine's effect on ethanol-induced amnesia and ethanol state-dependent memory was investigated. Adult male mice were cannulated in the CA1 regions of the dorsal hippocampus and trained on a passive avoidance learning task for memory assessment. We found that pre-training intraperitoneal (i.p.) administration of ethanol (1 g/kg) decreased inhibitory avoidance memory when tested 24 h later. The response induced by pre-training ethanol was significantly reversed by pre-test administration of the drug. Similar to ethanol, pre-test administration of nicotine (0.4 and 0.8 µg/mouse, intra-CA1) alone and nicotine (0.2, 0.4 and 0.8 µg/mouse) plus an ineffective dose of ethanol also significantly reversed the amnesia induced by ethanol. Ethanol amnesia was also prevented by pre-test administration of L-arginine (1.2 µg/mouse, intra-CA1), a NO precursor. Interestingly, an ineffective dose of nicotine (0.2 µg/mouse) in combination with a low dose of L-arginine (0.8 µg/mouse) synergistically improved memory performance impaired by ethanol given before training. In contrast, pre-test intra-CA1 microinjection of L-NAME (NG-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor (0.4 and 0.8 µg/mouse), which reduced memory retrieval in inhibitory avoidance task by itself, in combination with an effective dose of nicotine (0.4 µg/mouse) prevented the improving effect of nicotine on memory impaired by pre-training ethanol. Moreover, intra-CA1 microinjection of L-NAME reversed the L-arginine-induced potentiation of the nicotine response. The results suggest the importance of NO system(s) in the CA1 regions of the dorsal hippocampus for improving the effect of nicotine on the ethanol-induced amnesia.

In-vivo extravasation induces the expression of interleukin 1 receptor type 1 in human neutrophils.

In order to address neutrophil activation during inflammation we assessed the expression of interleukin 1 receptor type 1 (IL-1R1) following in-vivo extravasation. Extravasated neutrophils were collected from 11 healthy study subjects by a skin chamber technique and compared to neutrophils in peripheral blood. Expression of IL-1R1 was assessed by microarray, quantitative polymerase chain reaction (qPCR), Western blot, flow cytometry, enzyme linked immunosorbent assay (ELISA) and immunoelectron microscopy (iEM). IL-1R1 was induced following extravasation, demonstrated by both gene array and qPCR. Western blot demonstrated an increased expression of IL-1R1 in extravasated leucocytes. This was confirmed further in neutrophils by flow cytometry and iEM that also demonstrated an increased intracellular pool of IL-1R1 that could be mobilized by N-formyl-methionine-leucine-phenylalanine (fMLP). Stimulation of peripheral neutrophils with IL-1 resulted in transcription of NFκB and a number of downstream chemokines and the corresponding chemokines were also induced following in-vivo extravasation. The present results demonstrate that IL-1R1 is induced following extravasation and exists on the neutrophil surface, as well as in a mobile intracellular pool. Furthermore, neutrophils express functional IL-1R1 as demonstrated by the induction of chemokines following IL-1 stimulation. The results indicate a potential role for IL-1 in the activation of neutrophils at inflammatory sites.

The first study on enhanced photoresponsivity of ZnO-TiO2 nanocomposite thin films by anodic polarization.

The physical and photoelectrochemical properties of the anodized ZnO-TiO(2) thin films were investigated in this study. Impedance spectroscopy revealed a decrease in charge transfer resistance and Tafel plots determined enhancement of about 200 times in the exchange current (i(0)) after anodization at high positive potential. It was found from XPS analysis that after applying the potential of 5 V to the ZnO-TiO(2) photoelectrode, the lattice oxygen (O(2-)) in the thin film is oxidized to molecular oxygen and then, cations such as Zn(2+) can be solved in the basic electrolyte and passed to the solution. Moreover, according to AFM analysis it was observed that the surface of the samples has been grooved by applying anodic voltage resulting in an increase of effective surface of the film. A mechanism for describing the significant enhancement in the photoresponse of the anodized layer is proposed.

Mutant p53 reactivation by PRIMA-1MET induces multiple signaling pathways converging on apoptosis.

The low molecular weight compound PRIMA-1(MET) reactivates mutant p53 and triggers mutant p53-dependent apoptosis in human tumor cells. We investigated the effect of PRIMA-1(MET) on global gene expression using microarray analysis of Saos-2 cells expressing His273 mutant p53 and parental p53 null Saos-2 cells. PRIMA-1(MET) affected transcription of a significantly larger number of genes in the mutant p53-expressing cells compared to the p53 null cells. Genes affected by PRIMA-1(MET) in a mutant p53-dependent manner include the cell-cycle regulators GADD45B and 14-3-3gamma and the pro-apoptotic Noxa. Several of the affected genes are known p53 target genes and/or contain p53 DNA-binding motifs. We also found mutant p53-dependent disruption of the cytoskeleton, as well as transcriptional activation of the XBP1 gene and cleavage of its mRNA, a marker for endoplasmic reticulum stress. Our data show that PRIMA-1(MET) induces apoptosis through multiple transcription-dependent and -independent pathways. Such integral engagement of multiple pathways leading to apoptosis is consistent with restoration of wild-type properties to mutant p53 and is likely to reduce the risk of drug resistance development in clinical applications of PRIMA-1(MET).

Identifying sources of reporting error using measured food intake.

OBJECTIVE: To investigate the magnitude and relative contribution of different sources of measurement errors present in the estimation of food intake via the 24-h recall technique. DESIGN: We applied variance decomposition methods to the difference between data obtained from the USDA's Automated Multiple Pass Method (AMPM) 24-h recall technique and measured food intake (MFI) from a 16-week cafeteria-style feeding study. The average and the variance of biases, defined as the difference between AMPM and MFI, were analyzed by macronutrient content, subject and nine categories of foods. SUBJECTS: Twelve healthy, lean men (age, 39+/-9 year; weight, 79.9+/-8.3 kg; and BMI, 24.1+/-1.4 kg/m2). RESULTS: Mean food intakes for AMPM and MFI were not significantly different (no overall bias), but within-subject differences for energy (EI), protein, fat and carbohydrate intakes were 14, 18, 23 and 15% of daily intake, respectively. Mass (incorrect portion size) and deletion (subject did not report foods eaten) errors were each responsible for about one-third of the total error. Vegetables constituted 8% of EI but represented >25% of the error across macronutrients, whereas grains that contributed 32% of EI contributed only 12% of the error across macronutrients. CONCLUSIONS: Although the major sources of reporting error were mass and deletion errors, individual subjects differed widely in the magnitude and types of errors they made.

Preserved leukocyte CD11b expression at the site of interstitial inflammation in patients with high-flux hemodiafiltration.

The impact of high-flux hemodialysis on clinical outcomes remains controversial. We have previously shown that in vivo transmigrated leukocytes from patients with low-flux bioincompatible hemodialysis have an impaired capacity to upregulate CD11b at the site of interstitial inflammation. In the present study, we investigated the in vivo capacity of transmigrated monocytes and granulocytes to express CD11b at the site of interstitial inflammation in 10 patients on biocompatible high-flux hemodiafiltration or high-flux hemodialysis and 12 healthy subjects, and the in vitro response to a bacteria-related peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)). Leukocyte formation of hydrogen peroxide (H(2)O(2)) and leukocyte apoptosis were also studied. In patients, both monocytes and granulocytes had a preserved capacity to express CD11b following in vivo transmigration to sites of interstitial inflammation, compared with cells from healthy subjects. Furthermore, monocytes and granulocytes from patients showed a preserved ability to respond to challenge with fMLP in the extravascular milieu. The intracellular killing capacity of leukocytes (H(2)O(2) production) in the interstitium was similar as of cells from healthy subjects both before and after stimulation with fMLP. Following maximal receptor independent stimulation (phorbol 12-myristate 13-acetate), leukocytes from patients showed lower H(2)O(2) production at the site of intense inflammation, compared with cells from healthy subjects. Finally, leukocyte apoptosis in interstitial inflammation was similar in patients and healthy subjects. We conclude that in vivo transmigrated leukocytes from patients on biocompatible high-flux hemodiafiltration or high-flux hemodialysis have a preserved capacity to express CD11b at the site of interstitial inflammation. This may have important biological implications.

Apoptotic tumor cells are superior to tumor cell lysate, and tumor cell RNA in induction of autologous T cell response in B-CLL.

B cell chronic lymphocytic leukemia (B-CLL) is a chronic leukemia manifested by increased numbers of B cells in circulation. The slow, smouldering nature of the disease in a significant proportion of the cases makes it an ideal target for immunotherapy. Dendritic cell (DC)-based immunotherapy is emerging as an exciting modality with significant clinical potential. In this study, three strategies for delivering antigens to DC, namely apoptotic bodies (Apo-DC), tumor lysates, and tumor RNA were studied in an autologous setting. In all six CLL patients, Apo-DC induced higher HLA-restricted, T cell responses than DC pulsed with tumor lysate or RNA. Real-time PCR confirmed higher expression of genes for IL-2 and IFN-gamma in T cells stimulated with Apo-DC. Concurrently, no IL-10 and low IL-4 responses indicated that the immune response was primarily of the Th1 type. Enzyme-linked immunospot assay revealed high IFN-gamma secretion by T cells when Apo-DC was used to stimulate autologous T cells in all patients. Our data suggest that cellular vaccines with DC loaded with apoptotic bodies may be a suitable approach for immunotherapy of B-CLL.

Lung accumulations of eosinophil granulocytes after exposure to cornstarch glove powder.

Starch is a main component of wheat flour, which, besides being an occupational allergen can also induce irritative symptoms in the airways. A purified starch product (cornstarch glove powder) was used to investigate whether starch alone could induce airway inflammation. The aim of the study was to investigate a role for starch in wheat flour-induced airway inflammation. Ten healthy individuals were exposed to cornstarch glove powder in a whole-body exposure chamber. Bronchoscopy with bronchoalveolar lavage (BAL) was performed 2-3 weeks before and 1 day after exposure, and the BAL cells were counted differentially. In addition, the expression of activation, adhesion and subset markers on alveolar macrophages and BAL T-cells were investigated using flow cytometry. A three-fold increase in BAL cell concentrations was found, with a selective accumulation and activation of eosinophilic granulocytes, as well as an influx of nonactivated monocytes and polyclonal CD4+ T-cells into the airways. The results show that inhalation of cornstarch glove powder leads to the development of a subclinical inflammation in the airways, with an accumulation of eosinophilic granulocytes. The authors suggest that such exposure may be an interesting model for studying factors contributing to lung accumulations of eosinophil granulocytes in humans.

Increased eosinophil transmigration after nasal allergen challenge in children with allergic asthma and rhinitis.

BACKGROUND: Eotaxin and interleukin-5 together provide the signal essential for eosinophil transmigration to airway tissue in allergic reactions. However, it is not known whether peripheral blood eosinophils (PBE) possess an increased transmigration capacity in vitro after allergen challenge in vivo before they leave the circulation. We aimed to determine whether PBE in cat-sensitized children have increased spontaneous and/or eotaxin-induced transmigration capacity in vitro, and to what extent allergen challenge alters this feature. METHODS: Fourteen cat-allergic children and four healthy controls underwent nasal challenge with cat-allergen. Blood samples were drawn prechallenge and at 2 h and 24 h postchallenge. We analyzed the in vitro transmigration of PBE, with and without eotaxin as a chemoattractant. We used a transmigration assay with fibronectin-coated membranes. Eosinophil cationic protein (ECP) and PBE counts were run in parallel. RESULTS: The spontaneous transmigration capacity of eosinophils in vitro was significantly higher at 2 h after allergen challenge (P < 0.01 vs. prechallenge) and returned to prechallenge levels at 24 h postchallenge (P < 0.02 vs. 2 h postchallenge). Addition of eotaxin further augmented the increased transmigration. In concordance, no accompanying changes were measured in the levels of eosinophils in blood or ECP in serum. Furthermore no spontaneous or eotaxin-induced eosinophil transmigration was detected in healthy controls. CONCLUSION: PBE possess increased spontaneous (and eotaxin-induced) capacity to transmigrate as early as 2 h after allergen challenge in allergic children, without accompanying signs of eosinophil activation in terms of increased PBE count or ECP level. This is probably due to the increased stage of activation of the eosinophil, often referred to as "priming".

Distinct phenotypic adhesion molecule expression on human cord blood progenitors during early Eosinophilic commitment: upregulation of beta(7) integrins.

Increasing levels of proinflammatory cells, including eosinophils and basophils, are seen at the site of allergen challenge in allergic disease of the airways. Mechanisms for the recruitment of these cell types could involve either specific upregulation of adhesion molecule and chemoattraction, or the initiation of proliferation and differentiation of inflammatory cell progenitors derived from the bone marrow. In this study, we demonstrate, in two systems of eosinophilic-basophilic lineage-committed granulocytes of relative immaturity, that eosinophilic differentiation in vivo implies the induction of a distinct adhesion phenotype, characterized by the upregulation of beta(7) integrin and downregulation of beta(1) and alpha(5) integrins. Moreover, the eosinophilic differentiation induced an upregulation of complement receptor type 1 and type 3, and the expression was further enhanced upon a short-course in vitro activation with ionomycin. These data indicate a sequential alteration of disparate members of the integrin family during eosinophilic-basophilic differentiation, which may attribute to specific adhesion requirements at distinct stages of cell maturation.

Self-sensitized photooxygenation of 3,4-dialkoxyfurans to vitamin C or its derivatives.

Self-sensitized photooxygenation of 3,4-dialkoxyfurans 3a-d with molecular oxygen and UV- or sunlight at room temperature gave vitamin C derivatives 2a-d in good to excellent yields. Furan 3c, having photodegradable protecting groups, was also photooxygenated to give L-ascorbic acid (1) in a "one-pot" reaction. Furthermore, a novel photolytic transformation was developed for deuteration of furan 3b at the C-2 position with D(2)O to give furan 3d in 95% yield. Toxicity of furans 3a-c and butenolides 2a-c against human embryonic cell, murine embryo fibroblasts, normal fibroblasts, HeLa, and Vero cell lines in the presence of oxygen and indirect solar light was found to be much less than those of the antipsoriasis drugs anthralin and 8-methoxypsoralen.

Design, synthesis, and biological evaluation of novel nucleoside and nucleotide analogues as agents against DNA viruses and/or retroviruses.

A novel strategy was developed for the synthesis of N(7)-purine acyclic nucleosides 9 and 14. The key step involved the reaction between [2-(p-methoxyphenyloxy)ethoxy]methyl chloride and N(9)-tritylated nucleobases 6 or 11 followed by concomitant self-detritylation. N(7)-Guanine acyclic nucleoside 9 exhibited antiviral activity, but was phosphorylated by both HSV and Vero cell thymidine kinases. Thus, it showed more potent cellular toxicity than acyclovir (2). N(7)-Adenine acyclic nucleoside 14 was found to be an excellent antiviral agent as well as a good inhibitor of calf mucosal adenosine deaminase. This inhibitory property allows for a greater expression of antiviral activity of antiviral agents, such as N(9)-adenine acyclic nucleoside 1 and ara-A (3). Compound 14 was phosphorylated neither by herpes simplex virus (HSV) thymidine kinase nor by Vero cell thymidine kinase, yet it enhanced the rate constant for the monophosphorylation of acyclovir (2) by HSV thymidine kinase. Consequently, the combination of acyclovir (2) and 14 exhibited greater antiviral activity than acyclovir alone. 7-[2-(Phosphonomethoxy)ethyl]adenine (20) was also synthesized. The key step involved the reaction of 9-(2-cyanoethyl)adenine (15) with methyl iodoacetate in the presence of lithium 2,2,6,6-tetramethylpiperidine in THF. Unlike 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, 4), the N(7)-isomer 20 was not phosphorylated effectively by 5-phosphoribosyl 1-pyrophosphate synthetase (PRPP synthetase). Thus, it did not exhibit pronounced antiviral activity. Dinucleotide 5'-monophosphate 24 and its butenolide ester 25 were also synthesized. Compound 24 showed substrate activity toward PRPP synthetase and exhibited notable activity against DNA viruses. The antiviral activity of the ester derivative 25 was found to be higher than that of the parent molecule 24. Dinucleotide 5'-monophosphate 24 is susceptible to degradation by snake venom and spleen phosphodiesterases. However, its respective butenolide ester derivative 25 was completely resistant to snake venom and spleen enzymes. Butenolide ester derivatives 28 and 29 were also synthesized and exhibited notable anti-DNA virus and anti-retrovirus activity in vitro. Compounds 2, 4, 9, 14, 20, 24, 25, and 28 were also evaluated for their inhibitory effect on HSV-1-induced mortality in NMRI mice. N(7)-adenine acyclic nucleoside 14 [LD(50) (intraperitoneal, ip) 950 mg/kg], nucleotide-containing butenolide 25 [LD(50) (ip) 675 mg/kg], and butenolide 28 [LD(50) (ip) 710 mg/kg] were found to be potent anti-HSV-1 agents in vivo. In addition, butenolide 28 efficiently decreased tumor formation induced by Moloney murine sarcoma virus (MSV) in NMRI mice while significantly increasing the survival time of MSV-infected mice.

Effects of lexical factors on word recognition among normal-hearing and hearing-impaired listeners.

An investigation was conducted to examine the effects of lexical difficulty on spoken word recognition among young normal-hearing and middle-aged and older listeners with hearing loss. Two word lists, based on the lexical characteristics of word frequency and neighborhood density and frequency (Neighborhood Activation Model [NAM]), were developed: (1) lexically "easy" words with high word frequency and a low number and frequency of words phonemically similar to the target word and (2) lexically "hard" words with low word frequency and a high number and frequency of words phonemically similar to the target word. Simple and transformed up-down adaptive strategies were used to estimate performance levels at several locations on the performance-intensity functions of the words. The results verified predictions of the NAM and showed that easy words produced more favorable performance levels than hard words at an equal intelligibility. Although the slopes of the performance-intensity function for the hearing-impaired listeners were less steep than those of normal-hearing listeners, the effects of lexical difficulty on performance were similar for both groups.