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The objective of the study was to determine expression of gene of insulin receptor (INSR) in adipose tissue of postpartum dairy cows fed diets containing different amounts of energy at the antepartum period. Healthy pregnant dairy cows (n=45) on 21st d of the antepartum were divided into three groups differing in diet composition, namely: control group fed a normal diet, high energy group fed a high energy diet, and low energy group fed a low energy diet. Twenty-one days after parturition, INSR gene expression in adipose tissue was determined by internally controlled reverse transcriptase PCR. The level of INSR mRNA in adipose tissues of cows fed the high energy diet was substantially lower than that in cows fed normal or low energy diets. A relatively higher level of INSR mRNA in the adipose tissue of cows fed low energy diet may be beneficial for gluconeogenesis and lipogenesis, which can relieve an energy negative balance. Reduced level of INSR mRNA in adipose tissue of cows fed high energy diet indicates that the response to insulin has significantly decreased.

In recent years, there has been increasing interest in using LC-MS/MS to measure protein drugs (biotherapeutics) in plasma/serum to support pharmacokinetic/toxicokinetic (PK/TK) studies. A number of strategies have been proposed to quantify different types of protein drug candidates in biological matrices. However, the application of LC-MS/MS in biotherapeutics is still very limited in regulated bioanalysis due to practical challenges. In this manuscript, we propose a "data-dependent" approach for the LC-MS/MS support of protein drug candidates, focusing on operational aspects. We have developed and validated a fast, simple and reliable LC-MS/MS method to quantify a protein drug candidate in mouse serum. This method can fit into our current workflow for routine small molecule LC-MS/MS assays with an overall sample preparation time of less than two hours. The method has been demonstrated to be accurate, precise and rugged, and it has been successfully applied to analyze samples from toxicology studies. The results were verified using a ligand binding assay and tested by standard-addition sample reanalysis. In addition, general recommendations for developing and validating an LC-MS/MS assay based on surrogate peptide(s) of protein drug candidates are also proposed for regulated bioanalysis.

Strontium stimulates cartilage matrix formation in vitro. However, the mechanisms governing these effects have not yet been extensively reported. In this study, chondrocytes were isolated from rat articular cartilage by enzymatic digestion and cultured for 24-72 h with 1-5 mM strontium. We investigated the effects of different concentrations of strontium on collagen content, type II collagen, insulin-like growth factor (IGF-1) and matrix metalloproteinase (MMP)-13 expression in rat cultured articular chondrocytes in vitro. The collagen content of the chondrocytes, determined as hydroxyproline, was measured by a colorimetry method. Type II collagen, IGF-1, and MMP-13 mRNA abundance and protein expression levels were determined by real-time polymerase chain reaction (real-time PCR) and western blot, respectively. The results showed that collagen content from the chondrocytes extracellular matrix increased with increasing strontium concentration. Moreover, 3 and 5 mM strontium strongly stimulated protein expression and mRNA levels of type II collagen and IGF-1. Conversely, MMP-13 expression in chondrocytes decreased dose-dependently with increasing strontium concentration. These results should provide insight into the ability of strontium to promote chondrocyte extracellular matrix synthesis. Strontium could promote collagen synthesis and suppress collagen degradation via the repression of MMP-13 expression.

Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB) during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L) on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL), carnitine palmitoyltransferase IA (CPT IA), long chain acyl-CoA dehydrogenase (ACADL), and acetyl-CoA carboxylase (ACC). Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

RATIONALE Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents. METHODS An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity. RESULTS The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9. CONCLUSIONS By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey. Copyright © 2012 John Wiley & Sons, Ltd.

Dairy cows with ketosis are characterized by oxidative stress, hepatic damage, and hyperketonemia. Acetoacetate (AA) is the main component of ketone bodies in ketotic cows, and is associated with the above pathological process. However, the potential mechanism was not illuminated. Therefore, the aim of this study was to investigate the mechanism of AA-induced hepatic oxidative damage in ketotic cows. Compared with healthy cows, ketotic cows exhibited severe oxidative stress and hepatic damage. Moreover, the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of bovine hepatocytes. In this process, AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit nuclear factor E2-related factor 2 (Nrf2) expression, nuclear localization, and DNA-binding affinity, thereby inducing the overexpression of pro-apoptotic molecules Bax, Caspase 3, Caspase 9, PARP and inhibition of anti-apoptotic molecule Bcl-2. Antioxidant N-acetylcysteine (NAC) treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, these results indicate that AA triggers hepatocytes apoptosis via the ROS-mediated MAPKs pathway in ketotic cows.

Background: Creatinine is an endogenous compound generated from creatine by normal muscular metabolism. It is an important indicator of renal function and the serum level is routinely monitored in clinical labs. Results & methodology: Surrogate analyte (d3-creatinine) was used for calibration standard and quality control preparation and the relative instrument response ratio between creatinine and d3-creatinine was used to calculate the endogenous creatinine concentrations. Conclusion: A fit-for-purpose strategy of using a surrogate analyte and authentic matrix was adopted for this validation. The assay was the first human plasma assay using such strategy and was successfully applied to a clinical study to confirm a transient elevation of creatinine observed using an existing clinical assay.

Alpha-lipoic acid (ALA) has been reported to have beneficial effects for improving insulin sensitivity. However, the underlying molecular mechanism of the beneficial effects remains poorly understood. Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are considered causal factors that induce insulin resistance. In this study, we investigated the effect of ALA on the modulation of insulin resistance in ER-stressed HepG2 cells, and we explored the potential mechanism of this effect. HepG2 cells were incubated with tunicamycin (Tun) for 6h to establish an ER stress cell model. Tun treatment induced ER stress, mitochondrial dysfunction and insulin resistance. Interestingly, ALA had no significant effect on ER stress signals. Pretreatment of the ER stress cell model with ALA for 24h improved insulin sensitivity, restored the expression levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes and increased intracellular ATP production. Moreover, ALA augmented the β-oxidation capacity of the mitochondria. Importantly, ALA treatment could decrease oligomycin-induced mitochondrial dysfunction and then improved insulin resistance. Taken together, our data suggest that ALA prevents ER stress-induced insulin resistance by enhancing mitochondrial function.

This study was conducted to determine if adding the acid-tolerant engineered bacterial strain M. elsdenii H6F32 to the rumen of sheep would reduce the risk of lactic acidosis. Lactic acidosis were induced by feed challenges in three groups of six sheep intraruminally dosed with wheat. In each group, sheep were not supplemented(C) or supplemented with the original bacterial strain M.elsdenii H6(H6) or the acid-tolerant engineered bacterial strain M.elsdenii H6F32(H6F32). Compared with C, the concentrations of ruminal lactic acid were decreased and the pH were increased both in H6 and H6H32(P

β-Hydroxybutyrate (BHB) is an important indicator for metabolic disorders in dairy cows, such as ketosis and fatty liver. Dairy cows with ketosis display oxidative stress that may be associated with high levels of BHB. The purpose of this study was to demonstrate a correlation between the high levels of BHB and oxidative stress in dairy cows with ketosis, and to investigate the molecular mechanisms underlying oxidative damage in bovine hepatocytes. The results showed that dairy cows with ketosis exhibited oxidative stress and liver damage, which was significantly correlated with plasma BHB. Similarly, high concentrations of BHB increased the oxidative stress of cow hepatocytes in vitro, resulting in the phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK), which led to increased expression, nuclear localization, and transcriptional activity of p53 and decreased Nrf2 in bovine hepatocytes. High concentrations of BHB significantly increased the expression of proapoptotic genes and significantly inhibited the expression of antiapoptotic genes. Finally, high concentrations of BHB promoted apoptosis in bovine hepatocytes. N-Acetyl-l-cysteine, glucose, and SB203580 (p38 inhibitor) significantly attenuated BHB-induced apoptotic damage in hepatocytes. These results indicate that BHB induces bovine hepatocyte apoptosis through the ROS-p38-p53/Nrf2 signaling pathway.

Elevated levels of blood interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor–α (TNF–α) increase insulin resistance and result in inflammation. It is not clear whether elevated blood level of acetoacetate (ACAC) and decreased blood level of glucose, which are the predominant characteristics of clinical biochemistry in ketotic dairy cows, increase proinflammatory cytokines and subsequent inflammation. The objective of this study was to test the hypothesis that ACAC and glucose activate the NF-κB signalling pathway to regulate cytokines expression in bovine hepatocytes. Bovine hepatocytes were cultured with ACAC (0–4·8 mm) and glucose (0–5·55 mm) with or without NF-κB inhibitor PDTC for 24 h. The secretion and mRNA levels of cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The NF-κB signalling pathway activation was evaluated by western blotting. Results showed that the secretion and expression of IL-1β, IL-6 and TNF-α increased in an ACAC dose-dependent manner. Additionally, there was an increase in the secretion and mRNA expression of these three cytokines in glucose treatment group, which increased significantly when the glucose concentrations exceed 3·33 mm. Furthermore, both ACAC and glucose upregulated NF-κB p65 protein expression and IκBα phosphorylation levels. However, these effects were reduced by PDTC. These results demonstrate that elevated levels of ACAC and glucose increase the synthesis and expression of proinflammatory factors by activating NF-κB signalling pathway in hepatocytes, which may contribute to inflammation injury in ketotic dairy cows.

The objective of this study was to test the hypothesis that p-cymene can attenuate acute lung injury induced by lipopolysaccharide (LPS) in vivo. In the mouse model of LPS-induced acute lung injury, intraperitoneal preconditioning with p-cymene resulted in a significant reduction of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), lung water gain, inflammatory cell infiltration, lung tissue myeloperoxidase activity. In addition, p-cymene blocked the phosphorylation of IκBα protein and mitogen-activated protein kinases (MAPK) signaling pathway activation. Histopathologic examination of lung tissue indicated that p-cymene treatment markedly decreased focal thickening, congestion, pulmonary edema, and inflammatory cells infiltration. The results showed that p-cymene had a protective effect on LPS-induced ALI in mice.

Reflection coefficients play an important role in the quantitative evaluation of the bonding quality of the interfaces of the multi-layered composite material. This paper proposes a reflection coefficient-based echo signal model and a new Gaussian model based blind deconvolution method for extracting reflection coefficients of bonding interface. The traditional deconvolution problem can be converted into a parameters estimation process in this model. Moreover, in order to avoid the potential computational burden caused by multiple parameters estimation and the much dependence on the initial parameter values during the estimation process, empirical mode decomposition (EMD) method and an improved Genetic Algorithm are used for parameters estimation. Simulation experiment verified the reliability of this method at different signal-to-noise ratio and with different initial parameter values, and then, reflection coefficients reconstruction of a metal multi-layered composite material specimen is performed through measurement experiment and the result is satisfying. Both the simulation and measurement experiments proved the feasibility and validity of this method.

The transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity of Veillonellaceae organisms was reduced and that of Streptococcaceae organisms was increased in rumen samples from the transition period and ketosis groups, with the number of Lactobacillaceae organisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes, Megasphaera elsdenii and Selenomonas ruminantium , were decreased, while numbers of the main lactate-producing bacterium, Streptococcus bovis , were increased in the rumen of cows from the transition period and ketosis groups, with the number of Lactobacillus sp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers of Selenomonas ruminantium and Megasphaera elsdenii organisms in the rumen.

Primary hepatocytes are commonly used during in vitro studies, but care must be taken with isolation and culture of the cells to ensure their viability. In this study, hepatocytes were isolated from the liver (caudate process) of a newborn calf by the collagenase perfusion and digestion method. The trypan blue exclusion method was used to determine total cell number and the survival rate of hepatocytes, while hepatocyte function was assessed by measuring lactate dehydrogenase, albumin and urea in culture medium supernatants at 24, 48, 72, 96, 120, 144 and 168 h. Results showed that the number of viable cells/g of liver (wet weight) averaged 1.12×10(7) cells/g, with an average hepatocyte viability of 85.7% (range 83-92%). After 48 h of culture, the hepatocytes solidly adhered to the well culture plate and were spread in an epithelioid shape, with clear cell boundaries between the cells and biliary ductule-like structures formed which persisted for up to 10 days. Hepatocyte function was optimal at 72 h after isolation and culture. This simple and economical procedure for the isolation and culture of viable cells may be useful for in vitro bovine hepatocyte studies.

The objective of our study was to evaluate whether feeding pseudopurpurin affects bone mineral density and bone geometry architecture in rats. Pseudopurpurin was extracted, analyzed and purified using an HLPC-ESI-MS. Rats were given 0% and 0.5% pseudopurpurin powder in their diet. Femurs of rats were examined at 0.5, 1 and 2 months after pseudopurpurin feeding. Compared with rats in the group 0%, the bone mineral density, and the calcium, magnesium, zinc and manganese concentrations in the rats femur in the group 0.5% increased significantly at 1 month and 2 months after pseudopurpurin feeding. Analytical results of micro-computed tomography showed that the group 0.5% displayed an increase in the trabecular volume fraction, trabecular thickness and trabecular number of the distal femur at 1 and 2 months after pseudopurpurin feeding, and the mean thickness, inner perimeter, outer perimeter, and area of the femur diaphysis were significantly increased at 2 months after pseudopurpurin feeding compared with the group 0%. In parallel, the trabecular separation and structure model index of the distal femur were decreased, compared with the group 0% at 1 and 2 months after pseudopurpurin feeding. In the 0.5% and 0% groups, there was no damage to kidney and liver by histopathology analysis. The long-term feeding of pseudopurpurin is safe for rats. The feeding of 0.5% pseudopurpurin which has specific chemical affinities for calcium for bone improvement and level of bone mineral density, enhances the geometry architecture compared with the 0% group.

The experiment was performed to evaluate the influence of copper supplementation on insulin-like growth factor-1 (IGF-1) mRNA expression in chondrocytes of newborn pigs. Chondrocytes were isolated and cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5 μmol/L copper in 90-mm culture plate. After 0, 12, 24, and 48 h, total RNA was isolated from chondrocytes. Then, IGF-1 mRNA expression was determined by semiquantitative RT-PCR. The results showed that the expression of IGF-1 mRNA, adjusted for β-actin expression, was increased in the culture media added to 15.6, 31.2, and 62.5 μmol/L copper, respectively. In the present experiment, the optimal copper concentration and optimal culture time for the expression of IGF-1 mRNA were 31.2 μmol/L and 48 h, respectively.

Phosphatase and tensin homolog (PTEN) is a potent tumor suppressor gene that also plays a vital role in regulating fatty acid metabolism. Here we attempted to elucidate the role of PTEN in the regulation of fatty acid oxidation and the assembly and secretion of very low density lipoprotein (VLDL) in dairy cow liver.We transfected primary culture calf hepatocytes with adenovirus-mediated PTEN overexpression vector (AD-GFP-PTEN).PTEN-overexpressing hepatocytes and control hepatocytes were obtained.Compared with controls, overexpression of PTEN significantly up-regulated CPT I, ACSL, HADH expression (p0.05), which are all involved in fatty acid oxidation. At the same time, the expression of ApoB100 (p0.01), ApoE (p0.05) and MTP (p0.01) increased. Therefore, the assembly and secretion of VLDL was enhanced (p0.05). The expression of LDLR was slightly up-regulated, but there was no significant difference (p0.05). To demonstrate that fatty acid metabolism was changed, we measured the concentrations of TG and VLDL. The concentration of TG was significantly decreased in hepatocytes (p0.01), while the concentration of VLDL was significantly increased in the medium (P0.05).Overexpressing PTEN enhanced fatty acid oxidation and assembly and secretion of VLDL. PTEN gene therapy could have therapeutic potential for fatty liver diseases of dairy cattle.

As an uncommon multisystem inflammatory disease, Behçet's syndrome is characterized by mouth and genital ulcers, skin lesions, and eye inflammation, which may also affect joints, blood vessels, central nervous system, or digestive tract. However, the inflammation of coronary artery is relatively rare. We thereby reported a young male of Behçet's syndrome presenting left anterior descending artery pseudoaneurysm with myocardial infarction. Surgical resection was performed with coronary bypass grafting.

BALB/c mice were immunized four times with formalin-prepared abrin-a. Using the polyethylene glycol method, immunized splenocytes were isolated and fused with SP2/0 cells. An indirect ELISA was established and used to detect positive clones secreting monoclonal antibodies (mAbs) against abrin-a. After analysis, three hybridoma clones secreting IgG-subtype mAbs were obtained. The antibodies were purified from the hybridoma growth medium using protein A or G affinity chromatography. Western blot analysis was used to analyze the antigenic epitopes on abrin-a recognized by the mAbs. The mAbs were specific for abrin-a, with no detectable cross-reactivity with several homologous toxins and associated agglutinins. Sandwich ELISA was then developed using these mAbs, which had a detection limit for abrin-a of 7.8 ng/mL.

Dairy cows experience an increased demand for glucose to support milk production. However, negative energy balance is a common condition in peripartum cows. In response, fat mobilization provides non-esterified fatty acids (NEFAs) for oxidation in the liver to generate ATP. To investigate the effects of NEFAs on gluconeogenesis, the expression and enzyme activity of pyruvate carboxylase (PC) and phosphoenolpyruvate carboxylase (PEPCK) in cultured bovine hepatocytes were evaluated by quantitative polymerase chain reaction and spectrophotometry, respectively. The results showed that PC and PEPCK mRNA levels were marked decreased when the NEFAs concentrations exceeded 0.5 and 1.5 mmol/l, respectively. The PC and PEPCK enzyme activity showed significantly decreased when the NEFAs concentrations exceeded 1.5 and 0.5 mmol/l, respectively. These findings indicate that high circulating levels of NEFAs inhibit hepatocyte gluconeogenesis, thereby promoting negative energy balance.

A strong piezoresistive effect of GaAs micro-structure which is based on high electron mobility transistor (HEMT) is reported in this paper. The GaAs HEMT is embedded in the root of the cantilever as the sensitive element in order to detect the deformation. The strain is simulated with the ANSYS software, and the maximum gauge factor is about 26,350, which is nearly a hundred times larger than that of piezoresistive silicon. The high gauge factor is not only due to the option of voltage bias, but also the combination of the piezoresistive and piezoelectric effect. The obtained results demonstrate that GaAs micro-structure based on HEMT can be suitable for high sensitive stress/pressure sensors.

Dried blood spot (DBS) technology is an emerging alternative for sample collection in bioanalysis. Dilution for DBS samples is a challenge due to its solid sample format. Currently, DBS samples requiring dilution were first extracted as regular samples and then diluted with extracted blank samples containing internal standard (IS). Since the dilution step is a volume-critical step, extra care has to be taken to achieve accurate dilution when dealing with limited volume extracted samples. Here, we introduce an alternative sample dilution for liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays using IS to track the dilution step. Dilution factor-adjusted IS working solution was first added to the sample requiring dilution before sample processing; subsequently, the processed sample was approximately diluted into the assay linear response range before LC/MS/MS analysis. We define this approach as "IS-tracked dilution". The advantage of this approach is that the diluting step is tracked by the IS and is no longer a volume-critical step. Another recognized challenge related to sample dilution is automatic sample dilution using a liquid handler. This "IS-tracked dilution" may also help address some of the challenges for automatic sample dilution of liquid samples. This new dilution approach was proven to be effective and convenient in both plasma assays and DBS assays using omeprazole as a probe compound.

Chondrocytes from the lateral trochlear ridge of the distal femur taken from 1-day-old piglets were cultured in medium supplemented with 0, 7.8, 15.6, 31.2, and 62.5 μmol/L copper. Insulin-like growth factor-1 (IGF-1) and IGF-binding protein 3 (IGFBP-3) levels in culture medium were determined by radioimmunoassay. DNA synthesis in chondrocytes was measured by tritiated thymidine ((3)H-TdR) incorporation. Proliferation-promoting activity and incorporation of (3)H-TdR in chondrocytes were increased in all culture media supplemented with copper and 15% fetal calf serum (FCS). The contents of IGF-1 and IGFBP-3 were also enhanced significantly in culture media containing 15% FCS and supplemented with copper at 15.6, 31.2, and 62.5 μmol/L. The optimal copper concentration for promoting chondrocyte proliferation and autocrine secretion of IGF-1 and IGFBP-3 was 31.2 μmol/L.

Immunoassays for heavy metals offer an alternative approach to traditional techniques for detection of mercury. In this study, a mercury-chelate was prepared with 1-(4-aminobenzyl) ethylenediamine-N,N,N',N'-tetraacetic acid (aminobenzyl-EDTA). The resulting complex was linked to keyhole limpet hemocyanin (KLH) or bovine serum albumin via the amino group and used as the immunizing antigen or detection antigen, respectively. BALB/c mice were immunized with KLH-aminobenzyl-EDTA-Hg and spleen cells from BALB/C mice were fused with Sp2/0 cells. One cell line (5F7) produced monoclonal antibodies with preferential selectivity and sensitivity for aminobenzyl-EDTA-Hg. This cell line had an affinity constant of 4.31 × 10(9) L/mol and its cross-reactivity (CR) with other metals was2%. The antibody was used for competitive indirect ELISA (CI-ELISA) for Hg(2+) measurements. The detection range was 0.087-790.4 μg/L and the lower limit of detection was 0.042 μg/L. The concentrations of mercury in environmental water samples obtained by CI-ELISA correlated well with graphite furnace atomic absorption spectrometry (GFAAS), and the mean recovery was 88.82% to 104.64%. These results indicate that this method could be used for monitoring mercury of water.

This experiment was conducted to measure the effect of copper supplementation on TGF-β gene expression in chondrocytes of newborn pigs. Chondrocytes were cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5 μmol/L copper in 90-mm culture plate. Total RNA was isolated from chondrocytes, and TGF-β cDNA was synthesized, amplified, and sequenced. The expression level of TGF-β was examined by reverse transcription polymerase chain reaction. The results showed that the sequence of the cloned TGF-β gene was 99.4% identical to that in GenBank. The expression of TGF-β increased in culture media added with final concentration of 15.6, 31.2, and 62.5 μmol/L copper. In this study, the optimal copper concentration and optimal culture time for the highest level of TGF-β expression were 31.2 μmol/L and 48 h, respectively.

The major role of insulin and the insulin receptor (InsR) in the liver is to mediate glucose uptake into hepatocytes to synthesize glycogen and to maintain blood glucose homeostasis. In this study, we investigated the effects of high insulin concentrations on InsR gene expression in calf hepatocytes cultured in vitro. After the cells were cultured for 72 h, insulin was added to the culture solution at final concentrations of 0, 1, 10, 100 or 1000 nM. InsR mRNA expression was determined by semi-quantitative RT-PCR. The results showed that InsR mRNA expression in hepatocytes, adjusted for β-actin expression, decreased dose dependently with increasing insulin concentration. InsR mRNA expression was similar at 1 and 10 nM insulin, but was significantly lower than that in the control. InsR expression was similar at 100 and 1000 nM insulin, but was significantly lower than that in the control, 1 and 10 nM insulin groups. These data suggest that high concentrations of insulin significantly repress InsR mRNA expression in calf hepatocytes, and this inhibition occurs in a dose-dependent manner. Further studies are needed to investigate the mechanisms underlying these effects of insulin.

BACKGROUND: Ivosidenib, a potent mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor, is being assessed in a phase 1 study of mIDH1 advanced hematologic malignancies (NCT02074839). We characterized the pharmacokinetics (PK) of ivosidenib in this population, and the effects of patient/disease characteristics and concomitant medications. METHODS: Ivosidenib was given in continuous 28-day cycles at 100 mg twice daily and 300 mg, 500 mg, 800 mg, and 1200 mg once daily (QD). Enrollment is complete; 258 patients received ≥1 ivosidenib dose (78 in escalation, 180 in expansion); samples were available from 253 patients (223 received ivosidenib 500 mg QD). Ivosidenib concentrations were determined using validated liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based methods. Population PK modeling was conducted using NONMEM software. The impact of demographics, renal and hepatic function, disease type, Eastern Cooperative Oncology Group (ECOG) performance status, and concomitant cytochrome P450 3A4 (CYP3A4) inhibitors/inducers and gastric acid reducers on ivosidenib PK was explored. RESULTS: Ivosidenib PK were best described using a 2-compartment model with first-order absorption, dose-dependent bioavailability, and a time-dependent change in relative bioavailability and clearance between Day 1 and steady state. Mean steady-state apparent clearance (CL/F) was 5.39 L/h (between-patient variability ~35%) and mean central volume of distribution (Vc/F) was 234 L (~47%). Less than dose-proportional bioavailability was observed, with a dose doubling translating to a ~40% increase in exposure. The moderate/strong CYP3A4 inhibitors voriconazole, fluconazole, and posaconazole were associated with 36%, 41%, and 35% reductions in CL/F, and hence 57%, 69%, and 53% increases in area under the plasma ivosidenib concentration-time curve (AUC), respectively (Figure 1). Baseline body weight had a significant impact on Vc/F. Low albumin at baseline and during treatment correlated with decreased CL/F and Vc/F. However, the effects of body weight and albumin did not appear to be clinically relevant. No effects of creatinine clearance or measures of liver function (alanine aminotransferase, aspartate aminotransferase, bilirubin, within the range studied) on ivosidenib CL/F were detected. Concomitant use of pantoprazole or famotidine did not affect ivosidenib CL/F. CONCLUSION: This population PK model of ivosidenib suggests that no dose adjustments are needed based on the range of patient and disease characteristics analyzed. Disclosures Le: Millennium: Patents & Royalties; Agios: Employment, Equity Ownership. Wada:Certara: Employment; Agios: Consultancy. Dai:Agios: Employment, Equity Ownership. Fan:Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Attar:Agios: Employment, Equity Ownership. Agresta:Agios: Employment, Equity Ownership. Yang:Agios: Employment, Equity Ownership.

Mutations in isocitrate dehydrogenase 2 (IDH2) promote AML pathogenesis through production of 2-hydroxyglutarate (2-HG). Enasidenib is an inhibitor of mutant IDH2 activity and induces the differentiation of IDH2-mutated leukemic blasts. In a phase I/II clinical trial, enasidenib monotherapy resulted in an overall response rate of 40% and median duration of response of 6 months in relapsed/refractory AML (Stein et. al. Blood 2017). We previously discovered that mutant IDH activity sensitizes AML cells to BCL-2 inhibition through accumulation of 2-HG (Chan et. al. Nature Medicine 2015). Pharmacologic inhibition of BCL-2 activity with venetoclax, a highly specific BH3 mimetic, preferentially targets IDH-mutated human AML cells. In a phase II clinical trial of venetoclax monotherapy, IDH-mutated relapsed/refractory AML patients had a response rate of 33% compared to 10% in IDH-wildtype patients (Konopleva et. al. Cancer Discovery 2016). Based on the above findings, we hypothesized that combination therapy with enasidenib and venetoclax may demonstrate superior anti-leukemic activity in comparison to single agents in the treatment of IDH2-mutant AML. However, given that the mechanism by which IDH mutations increase BCL-2 dependence is via 2-HG, reduction of 2-HG with enasidenib may antagonize venetoclax activity. As such, we further hypothesized that a sequential dosing schedule may be superior to concurrent dosing. To test our hypotheses, we conducted a preclinical study to evaluate the efficacy of monotherapy versus combination therapy in reducing the leukemic burden in three patient-derived xenograft (PDX) models of human IDH2-mutant AML. All three PDX models were derived from samples with co-occurring IDH2R140Q and NPM1c mutations. Engrafted animals were randomly assigned to one of six treatment arms (N=5 per arm): vehicle (arm 1), enasidenib alone (arm 2), venetoclax alone (arm 3), concurrent combination (arm 4), and sequential combinations (arms 5 and 6; see Figure 1 for details). Enasidenib and venetoclax were administered by oral gavage at a dose of 40 mg/kg twice a day and 100 mg/kg daily, respectively. Tumor burden was measured in bone marrow samples collected immediately prior to treatment and every 2 weeks during the 12-week treatment period. Concurrent combination treatment (arm 4) resulted in the greatest reduction in leukemia engraftment compared to all other treatment arms, including the sequential dosing arms, in two of the three models (#1 and #2, henceforth termed "responders"; Figure 2A). Although venetoclax monotherapy reduced engraftment in all three models, persistent disease above a threshold of 0.1% was detectable in 12 of 13 animals by flow cytometry (9 of 13 by ddPCR) after 12 weeks of treatment (Figure 2B). In contrast, disease was detectable in only 2 of 9 animals (0 of 9 by ddPCR) treated with concurrent therapy in responders. In the remaining model (#3, henceforth termed "non-responder"), combination therapy was not superior to venetoclax monotherapy but importantly, co-treatment with enasidenib did not antagonize venetoclax activity. Interestingly, enasidenib monotherapy increased expression of myeloid differentiation markers, CD15 and CD11b, only in responders, indicating that differentiation might be a precondition for responsiveness to concurrent therapy. We confirmed that the lack of response in non-responders was not due to selection of an IDH2 wildtype clone or failure to block 2-HG production by enasidenib. To gain insights into the mechanism by which enasidenib might enhance venetoclax sensitivity, we performed quantitative intracellular flow cytometry staining for the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 in leukemic cells collected after 12 weeks of treatment. Enasidenib monotherapy resulted in a significant decrease in BCL-2 expression in responders. The reduction in anti-apoptotic protein expression could potentiate mitochondrial priming and sensitization to venetoclax. In summary, our findings demonstrate that concurrent combination therapy with enasidenib and venetoclax is a promising therapeutic approach for IDH2-mutated AML. Responsiveness to combination therapy is associated with enasidenib-induced differentiation and reduction in anti-apoptotic protein expression. Our findings support ongoing and future clinical investigations in combination therapies with mutant IDH and BCL-2 inhibitors. Disclosures Cojocari: AbbVie Inc: Employment. Phillips:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. MacBeth:Celgene Corporation: Employment, Equity Ownership. Nicolay:Agios: Employment. Narayanaswamy:Agios: Employment. Ronseaux:Agios: Employment. Liu:Agios: Employment, Equity Ownership. Chan:AbbVie: Research Funding; Genentech: Research Funding; Celgene: Research Funding.

This experiment was conducted to examine the effect of dietary copper supplementation on somatostatin (SS) and growth hormone-releasing hormone (GHRH) mRNA expression levels in the hypothalami of growing pigs. A total of 45 crossbred pigs were randomly assigned to three groups of 15 pigs each; five replicates of three animals comprised each group. Pigs were allocated to diets that contained 5 mg/kg copper (control), 125 mg/kg copper sulfate, or 125 mg/kg copper methionine. At the end of the experiment, five pigs were selected at random from each group and slaughtered, and hypothalami were collected for determination of SS and GHRH mRNA expression levels. The results showed that the SS expression levels were lower and the GHRH levels were higher in pigs fed the diets with 125 mg/kg copper methionine (P

Matrix ion suppression/enhancement is a well-observed and discussed phenomenon in electrospray ionization mass spectrometry. Nonuniform matrix ion suppression/enhancement across different types of samples in an analytical run is widely believed to be well compensated for by using a stable isotope-labeled internal standard (SIL-IS) in bioanalysis using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Therefore, the risk of nonuniform matrix ion suppression/enhancement is usually deemed low when an SIL-IS is used. Here, we have identified, evaluated, and proposed solutions to control bioanalytical risks from nonuniform matrix ion suppression/enhancement even with an SIL-IS through a case study using omeprazole. Two lots of human blank urine were tested, and ion enhancement of about 500% for omeprazole was observed in one lot but not in the other. When a quadratic regression model had to be used, the assay failed the industry acceptance criteria due to unacceptable positive bias for the middle and high quality control (QC) samples. The failure was attributed to different extents of matrix ion enhancement between the standards (STDs) and QCs, which resulted in the misaligned results from the regression model. It was concluded that, for the same amount of drug, nonuniform ion enhancement for different types of samples (STD or QC) resulted in different ion intensities, therefore leading to different response behaviors (linear or nonlinear) at the mass spectrometer detector. A simplified mathematical model was used to evaluate the risk when unmatched response models occurred for different types of samples. A diagnostic factor Q (Q = X(ULOQ)(-A/B)) was proposed to monitor the risks, where X(ULOQ) is the upper limit of quantitation of the assay, A is the quadratic slope of the curve, and B is the linear slope of the curve. The potential maximum errors were estimated on the basis of the mathematical model for different scenarios, and Q values were given to control the risks under these conditions for bioanalysis using LC-MS/MS.

Background: As a frequent subclinical disease, fatty liver disease (FLD) is associated with a severe negative energy balance (NEB) during the early lactation period, and usually cause of economic loss to dairy farmers. Liver biopsy is the gold standard for the assessment of FLD. However, as an invasive procedure, liver biopsy has several limitations and such procedures are not readily available to dairy farmers. To further evaluate FLD in dairy cows, a FLD model of lactating sheep was developed by simulation of the state of negative energy balance (NEB).Materials, Methods & Results: Fourteen pregnancy thin-tail ewes were divided into control group (CG, n = 4), non-lamb restrained feeding group (NRG, n = 4) and single birth restrained feeding group (SRG, n = 6). After lambing, NRG and SRG ewe were received a feed restrained diet for 16 days. Liver biopsies and blood was collected on days 1, 4, 7, 10, 13, and 16, and biochemical parameters were analyzed. With restricted feeding and lactation administration, ewes in SRG showed increased liver fat concentrations (LFC) from days 4 post-administration and severe LFC was detected at day 13. Compared with CG, SRG sheep showed significant lower concentration of serum glucose (Glu) from days 7-13 and higher non-esterified fatty acid (NEFA) from days 4-16, β-hydroxybutyric acid (BHBA) from days 4-16, triglyceride from days 4-16, low-density lipoprotein cholesterol from days 4-16, lactate dehydrogenase (LDH) from days 13-16, aspartate aminotransferase (AST) at days 16. While, ewes in NRG showed normal LFC levels, and high concentration of serum Glu and insulin from days 4-16 were detected than CG and SRG ewes. With restricted feeding, ewes in NRG and SRG showed significant low level of revised quantitative insulin sensitivity check index from days 4-16 and high level of liver total cholesterol (TC) at day 16. Liver pathological characteristics showed LFC of NEB sheep was first detected around the liver portal area.Discussion: In this study, a model of FLD in lactating thin-tail sheep was developed by restricted feeding. Serum glucose concentrations were sharply decreased in SRG sheep，that was due to the large energy requirements for lactation and low energy provided by a restricted diet. While non-lactating NRG sheep demonstrated lower fat mobilization, which was considered to contribute to the high concentrations of serum glucose, as compared to SRG sheep. Meanwhile, in a state of NEB, oxaloacetic acid, which is generated by glycolysis and glycogenic amino acids, tends to be used for gluconeogenesis, that a generous amount of NEFA is incompletely oxidized to generate ketone body in SRG sheep, which is a major component of BHBA. Liver TC concentrations were significantly higher in NRG sheep than those in the SRG sheep, while liver triglyceride was significantly lower. The high level of liver TC in NRG sheep was considered to induce removal of triglyceride from the liver in the form of VLDL. Compared with CG sheep, although higher levels of liver TC were detected in SRG sheep on postpartum day 16, these levels were considered too low to induce significant depletion of triglycerides from the liver. In this study, the increase in serum AST and LDH was considered to cause by oxidative stress in mitochondria, and LDH concentrations was considered more sensitively than AST for LFC caused by NEB. Liver pathological characteristics showed that FLD caused by NEB had a major impact on reduced LFC, although no significant liver fibrosis was detected. While different from FLD caused by high-fat diet, TG was first accumulates around the hepatic lobules and LFC of NEB sheep was first detected around the liver portal area. It was considered that high concentrations of NEFA are prioritized for oxygenation in the liver portal area, which results in triglyceride accumulation.

2581Background: Mutant isocitrate dehydrogenase 1 (mIDH1) produces the oncometabolite D-2-hydroxyglutarate (2-HG). Ivosidenib (IVO) is a selective mIDH1 inhibitor under evaluation in patients with ...

2577Background: Mutant isocitrate dehydrogenase 1 (mIDH1) produces the oncometabolite D-2-hydroxyglutarate (2-HG). Ivosidenib (IVO; AG-120) is a targeted mIDH1 inhibitor under evaluation in an ongo...

Complete oxidation of fatty acid in the liver of ketotic cows was investigated. Serum non-esterified fatty acid (NEFA), beta-hydroxybutyric acid (BHBA) and glucose concentrations were measured using biochemical techniques. Carnitine palmitoyl transferase II (CPT II), 3-hydroxy acyl-CoA dehydrogenase (HAD) and oxaloacetic acid (OAA) concentrations in the liver were detected by ELISA. Serum glucose was lower in ketotic cows than controls (p

Ketosis in dairy cows can lead to poor reproductive success and decreased milk production. Since the serum concentrations of copper (Cu) and zinc (Zn) are closely associated with the health status of cows, we investigated whether serum concentrations of Cu and Zn differed in dairy cows with subclinical ketosis and healthy dairy cows. Blood samples of 19 healthy dairy cows and 15 subclinically ketotic dairy cows were collected from three farms, and the concentrations of β-hydroxybutyrate (BHBA), glucose, non-esterified fatty acids (NEFA), Cu, and Zn were determined. Subclinically ketotic dairy cows had significantly higher BHBA and NEFA levels (p < 0.01) and lower glucose (p < 0.01) than healthy dairy cows. Likewise, serum concentrations of Zn were significantly decreased (p < 0.05) in dairy cows with subclinical ketosis. There was no significant difference observed for serum Cu concentration between healthy and subclinically ketotic dairy cows. This study suggests that a decreased serum Zn concentration could be a cause of decreased reproductive performance in subclinically ketotic dairy cows.

The objective of this study was to understand changes of plasma metabolites, hormones, and mRNA level of cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK-C) in liver in spontaneous clinical ketosis; 10 clinically ketotic cows and 10 healthy cows were chosen from the same dairy farm. Eleven blood parameters and liver fat content were measured in all cows, and mRNA levels of PEPCK-C in liver were measured by semi-quantitative reverse transcription (RT) polymerase chain reaction (PCR). In ketotic cows, concentration of plasma glucose decreased (p 0.05). In addition, concentration of plasma insulin decreased (p 0.05), and mRNA level of PEPCK-C in liver increased (p

The design, fabrication and test of a novel MEMS inclinometer were described. This inclinometer was based on the piezoresistive detection method, and was fabricated by SOI process. The micro-structure of the inclinometer was a four vertical cantilever beams, a center mass with a rigid cylinder fixed on it. The piezoresistors fabricated on the beams were used to detect the deformation of the cantilevers caused by the gravity. A test system was designed in this paper, from the test results we found that this inclinometer had a sensitivity of 0.025mV/^o, and the test results and the theoretical results are in well agreement, the standard deviation is 0.43874.

The objective of the present study was to identify differences in the expression levels of liver proteins between healthy and ketotic cows, establish a liver metabolic interrelationship of ketosis and elucidate the metabolic characteristics of the liver during ketosis. Liver samples from 8 healthy multiparous Hostein cows and 8 ketotic cows were pooled by health status and the proteins were separated by two-dimensional-electrophoresis (2D-E). Statistical analysis of gels was performed using PDQuest software 8.0. The differences in the expression levels of liver proteins (p

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event – a full immersion bioanalytical week – specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 1 covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (hybrid LBA/LCMS and regulatory agencies’ inputs) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will also be published in volume 7 of Bioanalysis, issues 23 and 24, respectively.

When abrin-a was combined with several polyclonal antibodies (PAb), the detection limit could be increased. In this way, a monoclonal antibody (capture) and polyclonal antibody (detection) sandwich enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-PAb conjugate-based immunochromatographic assay for detection of abrin-a were developed. The ELISA had a detection limit of 3.9 ng/mL for abrin-a in standard solution and 7.8 ng/mL in soybean milk, and was more sensitive than polyclonal antibody (capture) and monoclonal antibody (detection) ELISA, which had a detection limit of 15.6 ng/mL. The test strip had a detection range of 50 to 500 ng/mL for abrin-a and a detection limit in standard solution or soybean milk samples of 50 ng/mL. However, the test strip had a reduced detection capability compared with a colloidal gold-monoclonal antibody conjugate-based immunochromatographic assay test strip, which had a lower detection limit of 10 ng/mL. The developed ELISAs and test strip show the specificity towards abrin-a and have no cross-reactivity towards abrin-b, -c, -d, ricin, or the agglutinins from either castor beans or rosary peas.

Fads3 is the third member of the fatty acid desaturase gene cluster; with at least eight evolutionarily conserved alternative transcripts (AT), having no clearly established function as are known for FADS2 and FADS1. Here we present identification of a novel Fads3 transcript in mice (Fads3AT9), characterize Fads3AT9 expression in mouse tissues and evaluate correlations with metabolite profiles. Total RNA obtained from mouse tissues is reverse-transcribed into cDNA and used as template for PCR reactions. Tissue fatty acids were extracted and quantified by gas chromatography. Sequencing analysis revealed complete absence of exon 2 resulting in an open reading frame of 1239 bp, encoding a putative protein of 412 aa with loss of 37 aa compared to classical Fads3 (Fads3CS). FADS3AT9 retains all the conserved regions characteristic of front end desaturase (cytochrome b5 domain and three histidine repeats). Both Fads3CS and Fads3AT9 are ubiquitously expressed in 11 mouse tissues. Fads3AT9 abundance was greater than Fads3CS in pancreas, liver, spleen, brown adipose tissue and thymus. Fads3CS expression is low in pancreas while Fads3AT9 is over ten-fold greater abundance. The eicosanoid precursor fatty acid 20:4n - 6, the immediate desaturation product of the Fads1 coded Δ5-desaturase, was highest in pancreas where Fads3CS is low. Changes in expression patterns and fatty acid profiles suggest that Fads3AT9 may play a role in the regulation and/or biosynthesis of long chain polyunsaturated fatty acids from precursors.

In recent years, the applicability of using LC-MS/MS as a complementary technique to traditional ligand binding assays in the absolute quantitation of therapeutic proteins in biologic matrix has been demonstrated. Protein quantitation workflow via LC-MS/MS is primarily based on a enzymatic digestion model and recent works seek to improve selectivity and sensitivity. This review focuses on recent innovations in this field and discusses the following in detail: the applicability of two-dimensional liquid chromatography and its use to improve sensitivity and alleviate matrix ion suppression; the use of derivatization agents after digestion to improve extraction and MS ionization efficiency; techniques to reduce excess protein background and their positive effects on sensitivity, selectivity, and extraction consistency; the application of immunoaffinity extraction of proteins to enrich the analyte(s) of interest while improving selectivity and sensitivity.

It is known that there is a local biosynthesis of estradiol (E2) in breast carcinoma. The steroidogenic enzymes involved in E2 formation are aromatase which transforms testosterone into E2 and androstenedione into estrone (E1) and reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs) which convert E1 into E2. Using immunocytochemistry, we have studied the expression of aromatase and the three reductive 17β-HSDs 17β-HSD types 1, 7 and 12 in 41 specimens of female human breast carcinoma and adjacent non-malignant tissues. These results were correlated with the estrogen receptor α (ERα) and β (ERβ), progesterone receptor, androgen receptor, CDC47 and c-erb B-2 expressions and with the tumor stages. Aromatase was found in 58%, 17β-HSD type 7 in 47% and 17β-HSD type 12 in 83% of the breast cancer specimens. The 17β-HSD type 1 could be detected in only one tumor. A significant correlation was observed between the aromatase, 17β-HSD type 7 and 17β-HSD type 12 expression, as well as between each of the two enzymes 17β-types 7 and 12 and the ERβ expression. The expression of 17β-HSD type 12 was significantly higher in breast carcinoma specimens than in normal tissue. There was also a significant association of CDC 47 expression with ERβ, AR and 17β-HSD type 12. The results indicate that aromatase, 17β-HSD type 7 and 17β-HSD type 12, but not 17β-HSD type 1, are commonly expressed in human breast cancer. Moreover, the high expression of both 17β-HSD type 12 and ERβ in breast carcinoma cells may play a role in the development and/or progression of breast cancer.

Hypoxia, an insufficient level of oxygen in the cell, occurs during normal activity and also in pathological conditions such as ischemia and tumorigenesis. Although many hypoxia-response genes have been identified, an understanding of the functional role for these genes in the living animal is lacking. Here we present a genome-wide study of gene expression changes during hypoxia and then functionally test a subset of these genes for roles in survival and recovery from hypoxia. We found 79 genes with increased mRNA levels when adult flies were treated with 0.5% O2 for 6 h. A subset of these genes had detectably increased levels in as short as 1 h of low-oxygen treatment. Mild hypoxia levels resulted in an increase in transcription levels for only 20 genes. Viability during hypoxia and recovery time from hypoxia-induced paralysis was examined in flies with a reduction in activity in hypoxia-response genes. The observed decreased viability and increased recovery time from paralysis in many of the lines demonstrate that the increased transcript levels seen after hypoxia are important for the response to low oxygen.

The female parts of hops (Humulus lupulus L.) show estrogenic effects as well as cancer chemopreventive potential. We analyzed the chemopreventive mechanism of hops by studying its antioxidative activities and its effect on the detoxification of a potentially toxic quinone (menadione). The detoxification enzyme quinone reductase [(NAD(P)H:quinone oxidoreductase, QR] protects against quinone-induced toxicity and has been used as a marker in cancer chemoprevention studies. Although the hop extract was only a weak quencher of free radicals formed from 1,1-diphenyl-2-picrylhydrazyl, it demonstrated strong QR induction in Hepa 1c1c7 cells. In addition, compounds isolated from hops including xanthohumol (XH) and 8-prenylnaringenin were tested for QR induction. Among these, XH was the most effective at inducing QR with a concentration required to double the specific activity of QR (CD value) of 1.7 +/- 0.7 microM. In addition, pretreatment of Hepa1c1c7 cells with XH significantly inhibited menadione-induced DNA single-strand breaks. The QR inhibitor dicumarol reversed the protective effect of XH against menadione-induced DNA damage. Because the expression of QR and other detoxifying enzymes is known to be upregulated by binding of the transcription factor Nrf2 to the antioxidant response element (ARE), the reporter activity mediated by ARE in HepG2-ARE-C8 cells was investigated after incubation with XH for 24 h. Under these conditions, XH increased ARE reporter activity in a dose-dependent manner. One mechanism by which XH might induce QR could be through interaction with Keap1, which sequesters Nrf2 in the cytoplasm, so that it cannot activate the ARE. Using LC-MS-MS, we demonstrated that XH alkylates human Keap1 protein, most likely on a subset of the 27 cysteines of Keap1. This suggests that XH induces QR by covalently modifying the Keap1 protein. Therefore, XH and hops dietary supplements might function as chemopreventive agents, through induction of detoxification enzymes such as QR.