High Substrate Levels Research Articles

Protein lysine acetylation does not contribute to the high rates of fatty acid oxidation seen in the post-ischemic heart

High rates of cardiac fatty acid oxidation during reperfusion of ischemic hearts contribute to contractile dysfunction. This study aimed to investigate whether lysine acetylation affects fatty acid oxidation rates and recovery in post-ischemic hearts. Isolated working hearts from Sprague Dawley rats were perfused with 1.2 mM palmitate and 5 mM glucose and subjected to 30 min of ischemia and 40 min of reperfusion. Cardiac function, fatty acid oxidation, glucose oxidation, and glycolysis rates were compared between pre- and post-ischemic hearts. The acetylation status of enzymes involved in cardiac energy metabolism was assessed in both groups. Reperfusion after ischemia resulted in only a 41% recovery of cardiac work. Fatty acid oxidation and glycolysis rates increased while glucose oxidation rates decreased. The contribution of fatty acid oxidation to ATP production and TCA cycle activity increased from 90 to 93% and from 94.9 to 98.3%, respectively, in post-ischemic hearts. However, the overall acetylation status and acetylation levels of metabolic enzymes did not change in response to ischemia and reperfusion. These findings suggest that acetylation may not contribute to the high rates of fatty acid oxidation and reduced glucose oxidation observed in post-ischemic hearts perfused with high levels of palmitate substrate.

Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Bone mineral density and fracture risk in adult patients with hypophosphatasia

SummaryIn adult hypophosphatasia (HPP) patients, elevated lumbar spine dual X-ray absorptiometry (DXA) values are associated with markers of disease severity and disease-specific fracture risk while femoral bone mineral density (BMD), being largely unaffected by the disease severity, may still be useful to monitor other causes of increased fracture risk due to low BMD.IntroductionHypophosphatasia (HPP) is a rare inherited metabolic disorder due to deficient activity of the tissue-nonspecific alkaline phosphatase (TNAP). Clinical manifestation in adult HPP patients is manifold including an increased risk for fractures, but data regarding clinical significance of DXA measurement and associations with fracture risk and disease severity is scarce.MethodsRetrospective single-center analysis of DXA scans in patients with confirmed HPP (documented mutation, clinical symptoms, low alkaline phosphatase activity). Further data evaluation included disease-related fractures, laboratory results (alkaline phosphatase, pyridoxalphosphate, phosphoethanolamine), and medical history.ResultsAnalysis included 110 patients (84 female, mean age of 46.2 years) of whom 37.3% (n = 41) were harboring two mutations. Average T-Score level at the lumbar spine was − 0.1 (SD 1.9), and mean total hip T-Score was − 1.07 (SD 0.15). Both lower ALP activity and higher substrate levels (pyridoxalphosphate and phosphoethanolamine) were significantly correlated with increased lumbar spine T-Score levels (p < 0.001) while BMD at the hip was not affected by indicators of disease severity. Increased lumbar spine BMD was significantly associated with an increased risk for HPP-related fractures, prevalent in 22 (20%) patients (p < 0.001) with 21 of them having biallelic mutations.ConclusionBMD in adult HPP patients is not systematically reduced. Conversely, increased lumbar spine BMD appears to be associated with severely compromised mineralization and increased risk for HPP-related fractures while BMD at the hip appears unaffected by indicators of disease severity, suggesting suitability of this anatomic location for assessing and discerning disorders with increased fracture risk owing to reduced BMD like osteoporosis.Trial registration numberGerman register for clinical studies (DRKS00014022)Date of registration02/10/2018 – retrospectively registered

Role of RF Magnetron Sputtering Power on Optical and Electrical Properties of ITO Films on Soda-Lime Glass Substrates

The optical and electrical properties of indium tin oxide (ITO) thin films grown on soda-lime glass substrates using a radio frequency (RF) magnetron sputtering technique were studied as a function of the sputtering RF power. Fixed 100 nm thickness of ITO films were deposited on the soda-lime glass substrates at 300 °C, using RF powers ranging between 50 to 150 W. The optical and electrical properties of the sputtered ITO films were characterized by Ultraviolet–Visible Spectroscopy (UV-Vis), Hall Effect Measurement and Atomic Force Microscope (AFM). Varying the substrate temperature and RF sputtering power affected surface roughness to decrease from 5.80, 4.59 and 3.46 nm while the resistivity also decreased from 18.73 x 10−4, 6.484 x 10−4 and 2.421 x 10−4 Ω.cm. Deposition of ITO thin films on soda-lime glass substrates by RF magnetron sputtering at 300 °C substrate temperature contributed to 74 to 79 % of optical transmittance in the 450 nm region. Results of this study suggested that a better performance of both optical transparency and electrical conductivity of ITO films can be achieved by operating at high temperature substrate and high level of sputtering power.

Analyzing transient respirometric data by analytical algorithm for Monod kinetic parameters.

This study aims to develop an analytical algorithm with oxygen update (Ou) data obtained from transient respirometric measurement. Based on Monod kinetics, this study formulates a novel two-phase analytical model for an oxygen uptake rate plot (OUR vs. Ou) obtained by respirometric techniques. The first phase is a hyperbolic equation relating to exogenous and endogenous respiration, while the second phase is a linear equation for endogenous respiration only. An algorithm was therefore developed to analyze four Monod parameters by locating the best phase-separating point at which the absolute average relative error (ARE) of OUR is minimized. An analysis using test data on acetate verified that the algorithm is capable of transient kinetic parameter estimation with an ARE below 5-10%. A sensitivity analysis on domestic wastewater coupled with a Monte Carlo simulation concluded that the kinetic test must be conducted at a relatively high initial substrate level (So/Xo ≧ 1 and So/Ks ≧ 10) for reliable parameter estimation. Moreover, it is crucial to conduct the kinetic test with sufficient and acclimated seed culture for the degradation of substrate. The results of this study can be used to develop an automatic transient kinetic analyzer with modern programmable respirometers.

Effects of sulfur dosage and inoculum size on pilot-scale thermophilic bioleaching of heavy metals from sewage sludge

Land application of sewage sludge has received significant attention in recent years but the presence of elevated heavy metals in the sludge limits its land application. The purposes of this study were to investigate the effects of sulfur dosage and inoculum size on the thermophilic bioleaching of heavy metals from sewage sludge in a pilot-scale bioreactor. The microbial communities in this thermophilic bioleaching process were also identified using real-time polymerase chain reaction (real-time PCR). The results showed that the oxidation of sulfur and metal solubilization decreased with the increasing sulfur dosage. When the sulfur dosage was greater than 2% (w/v), the sulfur oxidation and metal solubilization rates decreased, indicating that the thermophilic bioleaching was hindered by high levels of substrate. However, it was found that the efficiency of metal solubilization and solid degradation was increased with the increase of inoculum size in the range from 5% to 20%. At the end of bioleaching, the efficiency of Mn, Zn, Ni, Cu and Cr from the sewage sludge reached 73–100%, 51–60%, 38–52%, 17–43% and 1–38%, respectively, while SS and VSS were degraded by 33–48% and 47–67%, respectively. Based on the analysis of real-time PCR, Sulfobacillus acidophilus was observed to be the predominant species (13–67% of total bacteria), whereas the populations of Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus were accounted relatively low (<1%).

Hypophosphatasia in Adults: Clinical Spectrum and Its Association With Genetics and Metabolic Substrates

Background: Hypophosphatasia (HPP) is a rare metabolic bone disorder caused by mutations in the alkaline phosphatase (ALPL) gene, and characterized by low circulating alkaline phosphatase (ALP) levels and bone, muscle, dental and systemic manifestations. In this case series we investigate the clinical spectrum, genetic and biochemical profile of adult HPP patients from the University Hospitals Leuven, Belgium. Methodology: Adults with HPP were identified through medical record review. Inclusion criteria were: (1) age ≥ 16 yr; (2) consecutively low ALP levels not explained by secondary causes; (3) one or more of the following supporting criteria: biochemical evidence of elevated enzyme substrates; subtrochanteric fractures, metatarsal fractures or other typical clinical features; family history of HPP; a known or likely pathogenic ALPL mutation. Results: Nineteen patients met our inclusion criteria (n = 2 infantile, n = 6 childhood, n = 10 adult-onset HPP and one asymptomatic carrier). Fractures and dental abnormalities were the most reported symptoms. Fatigue was reported in n = 7/19 patients (37%), three of which had previously been misdiagnosed as having chronic fatigue syndrome and/or fibromyalgia. Empirical pyridoxine therapy in four patients (without seizures) did not provide symptomatic relief. N = 7/19 patients (37%) were inappropriately treated or planned to be treated with antiresorptive treatment. Two patients developed atypical femoral fractures following exposure to bisphosphonates and/or denosumab. Patients detected by screening were less severely affected, while patients with homozygous or compound heterozygous mutations had the most severe symptoms, significantly lower circulating ALP levels (p = 0.013) and significantly higher pyridoxal-5’-phosphate (p = 0.0018) and urinary phosphoethanolamine (p = 0.0001) concentrations. Conclusions: Screening may detect mainly less severely affected individuals, which may nevertheless avoid misdiagnosis and inappropriate antiresorptive drug exposure. Patients with biallelic mutations had more severe symptoms, significantly lower ALP and higher substrate levels. Whether the latter finding has implications for the classification and treatment of HPP should be investigated further in larger cohorts.

Proanthocyanidin accumulation and transcriptional responses in the seed coat of cranberry beans (Phaseolus vulgaris L.) with different susceptibility to postharvest darkening

BackgroundEdible dry beans (Phaseolus vulgaris L.) that darken during postharvest storage are graded lower and are less marketable than their non-darkened counterparts. Seed coat darkening in susceptible genotypes is dependent upon the availability of proanthocyanidins, and their subsequent oxidation to reactive quinones. Mature cranberry beans lacking this postharvest darkening trait tend to be proanthocyanidin-deficient, although the underlying molecular and biochemical determinants for this metabolic phenomenon are unknown.ResultsSeed coat proanthocyanidin levels increased with plant maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these metabolites were absent in seeds of the non-darkening RIL plants. RNA sequencing (RNA-seq) analysis was used to monitor changes in the seed coat transcriptome as a function of bean development, where transcript levels were measured as fragments per kilobase of exon per million fragments mapped. A total of 1336 genes were differentially expressed between darkening and non-darkening cranberry bean RILs. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were upregulated in seed coats of the darkening RIL. A principal component analysis determined that changes in transcript levels for two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and ANTHOCYANIDIN REDUCTASE 1 (PvANR1) were highly correlated with proanthocyanidin accumulation in seed coats of the darkening-susceptible cranberry bean RIL. HPLC-DAD analysis revealed that in vitro activity of a recombinant PvANR1 was NADPH-dependent and assays containing cyanidin yielded epicatechin and catechin; high cyanidin substrate levels inhibited the formation of both of these products.ConclusionProanthocyanidin oxidation is a pre-requisite for postharvest-related seed coat darkening in dicotyledonous seeds. In model plant species, the accumulation of proanthocyanidins is dependent upon upregulation of biosynthetic genes. In this study, proanthocyanidin production in cranberry bean seed coats was strongly associated with an increase in PvANR1 transcripts during seed maturation. In the presence of NADPH, PvANR1 converted the physiologically relevant substrate cyanidin to epicatechin and catechin.

High dietary 18:2n-6/18:3n-3 ratio does not inhibit elongation and desaturation of 18:3n-3 to EPA and DHA in Atlantic salmon (Salmo salarL.)

This study investigated whether retention of n-3 fatty acids (FAs) is influenced by the levels of dietary monounsaturated (MUFA)-, saturated (SFA)- and n-6 FAs. The feeding trial used a mixture design, with rapeseed-, palm (PO)- and soybean oil (SO), providing high levels of MUFA, SFA and n-6 FAs, respectively, while 18:3n-3, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) were kept constant. Furthermore, a diet group with high SO and reduced 18:3n-3 was included in addition to the mixture design. There were no differences in growth or proximate composition, but the PO diet gave reduced FA digestibility and increased feed conversion ratio. All diet groups had a net production of DHA, shown by retention values >100% (133%–177%). High dietary n-6 FAs of up to 43% of FAs (18:2n-6/18:3n-3 ratio 8.6) did not negatively affect DHA retention, but rather had a small positive impact. High levels of substrate (dietary 18:3n-3 at ~10% of FAs) did not enhance DHA retention compared to 5% 18:3n-3. This is a highly significant finding for the aquaculture industry, allowing for greater flexibility in the choice of lipid sources to replace fish oil in salmon feeds.

Identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel autophagy regulator by high content shRNA screening.

Deregulation of autophagy has been linked to multiple degenerative diseases and cancer, thus the identification of novel autophagy regulators for potential therapeutic intervention is important. To meet this need, we developed a high content image-based shRNA screen monitoring levels of the autophagy substrate p62/SQSTM1. We identified 186 genes whose loss caused p62 accumulation indicative of autophagy blockade, and 67 genes whose loss enhanced p62 elimination indicative of autophagy stimulation. One putative autophagy stimulator, PFKFB4, drives flux through pentose phosphate pathway. Knockdown of PFKFB4 in prostate cancer cells increased p62 and reactive oxygen species (ROS), but surprisingly increased autophagic flux. Addition of the ROS scavenger N-acetyl cysteine prevented p62 accumulation in PFKFB4-depleted cells, suggesting that the upregulation of p62 and autophagy was a response to oxidative stress caused by PFKFB4 elimination. Thus, PFKFB4 suppresses oxidative stress and p62 accumulation, without which autophagy is stimulated likely as a ROS detoxification response.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Performance of Luffa cylindrica as immobilization matrix in bioconversion reactions by Nicotiana tabacum BY-2

The dry fruit of Luffa cylindrica was investigated as an immobilization matrix for Nicotiana tabacum cells in bioconversion reactions of exogenous substrates. Immobilized cells show high biocatalytic activity under high substrate levels. Cell growth on the dry fruit can be maintained until reaching an immobilization capacity of 1.8 g cells/g(Luffa).

Abstract 3395: BCR-Abl independent, Abl-TKI-therapy-resistant CML cells show enhanced sensitivity to GDC0941.

Abstract Introduction: Chronic Myeloid leukemia (CML) is driven by the BCR-ABL oncogene and is initially treated with ABL tyrosine-kinase inhibitors (ABL-TKI), such as Imatinib or Dasatinib. Despite strong initial responses to these drugs, many patients acquire resistance over time through acquisition of BCR-ABL mutations such as T315I, compound BCR-ABL mutations or through BCR-ABL-independent mechanisms. Even though the next generation pan-ABL inhibitor, Ponatinib, targets all know resistant ABL mutations, some patients don't benefit from Ponatinib and in these cases resistance to TKI therapy may be due to BCR-ABL-independent mechanisms. We analyzed the mechanism of ABL-TKI-dual-resistant CML cells independent of new BCR-ABL mutations and tested the sensitivity of these resistant cells to the PI3K inhibitor, GDC-0941 and/or the MEK inhibitor GDC-0973. Methods: We created ABL-TKI-dual-resistant cells and clones through prolonged treatment of K562 and KCL22 cells with Imatinib and then Dasatinib. The cells and clones were tested for sensitivity to Imatinib, Dasatinib, Ponatinib, GDC-0941 and GDC-0973. Whole genome copy number scan (Oncoscan) and targeted deep sequencing using GAII were used to identify BCR-ABL mutations and newly acquired somatic gene alterations. Phosphoproteomic analysis of cell lysates by Reverse Phase Protein Arrays was used to profile cell signaling pathway status associated with resistance and sensitivity to these drugs. Results: The K562 ABL-TKI-dual-resistant cells were insensitive to all three ABL-TKIs including Ponatinib suggesting that resistance was mediated by a BCR-ABL-independent mechanism. Unexpectedly, the resistant cells and clones became more sensitive to GDC-0941 but not GDC-0973 compared to the parental cells. These resistant cells and clones had acquired new somatic mutations in p53, BRCA2, PTEN, RB, SMARCA4 and PBRM1, but not in BCR-ABL. Phosphoprotein profiling showed low phosphorylation of the BCR-ABL substrates c-ABL, SHC and FAK indicative of BCR-ABL-independent mechanisms of resistance. Activity of the PI3K/AKT and MEK/ERK pathways varied across the resistant clones. However, high levels of the AKT substrate, FOXO-1 directly correlated with Dasatinib resistance and with GDC-0941 sensitivity. Sensitivity to GDC-0941 also correlated with modulation of phospho-FOXO-1. Our data suggest that GDC-0941 may be a therapeutic candidate for CML patients who progress on TKI therapy through BCR-ABL-independent mechanisms of resistance. Citation Format: Marie Wagle, Matthew Wongchenko, Shan Lu, Yinghui Guan, Yulei Wang, Mark Lackner, Garret Hampton, Yibing Yan. BCR-Abl independent, Abl-TKI-therapy-resistant CML cells show enhanced sensitivity to GDC0941. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2013-3395

Paclitaxel Resistance and Multicellular Spheroid Formation Are Induced by Kallikrein-Related Peptidase 4 in Serous Ovarian Cancer Cells in an Ascites Mimicking Microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Systematic Evaluation of Key L-Carnitine Homeostasis Mechanisms during Postnatal Development in Rat

BackgroundThe conditionally essential nutrient, L-carnitine, plays a critical role in a number of physiological processes vital to normal neonatal growth and development. We conducted a systematic evaluation of the developmental changes in key L-carnitine homeostasis mechanisms in the postnatal rat to better understand the interrelationship between these pathways and their correlation to ontogenic changes in L-carnitine levels during postnatal development.MethodsmRNA expression of heart, kidney and intestinal L-carnitine transporters, liver γ-butyrobetaine hydroxylase (Bbh) and trimethyllysine hydroxylase (Tmlh), and heart carnitine palmitoyltransferase (Cpt) were measured using quantitative RT-PCR. L-Carnitine levels were determined by HPLC-UV. Cpt and Bbh activity were measured by a spectrophotometric method and HPLC, respectively.ResultsSerum and heart L-carnitine levels increased with postnatal development. Increases in serum L-carnitine correlated significantly with postnatal increases in renal organic cation/carnitine transporter 2 (Octn2) expression, and was further matched by postnatal increases in intestinal Octn1 expression and hepatic γ-Bbh activity. Postnatal increases in heart L-carnitine levels were significantly correlated to postnatal increases in heart Octn2 expression. Although cardiac high energy phosphate substrate levels remained constant through postnatal development, creatine showed developmental increases with advancing neonatal age. mRNA levels of Cpt1b and Cpt2 significantly increased at postnatal day 20, which was not accompanied by a similar increase in activity.ConclusionsSeveral L-carnitine homeostasis pathways underwent significant ontogenesis during postnatal development in the rat. This information will facilitate future studies on factors affecting the developmental maturation of L-carnitine homeostasis mechanisms and how such factors might affect growth and development.

Characteristics of an aerobic denitrifier that utilizes ammonium and nitrate simultaneously under the oligotrophic niche

An aerobic denitrifier was isolated from the Hua-Jia-Chi pond in China and identified as Pseudomonas mendocina 3-7 (Genbank No. HQ285879). This isolated strain could express periplasmic nitrate reductase which is essential for aerobic denitrification occurred when the dissolved oxygen (DO) level maintains at 3-10 mg L(-1). To determine whether the ability of isolated strain is exhibited in the bioremediation of polluted drinking source water, the heterotrophic nitrification and aerobic denitrification characteristics of P. mendocina 3-7 under different cultural conditions such as oxygen level, nitrate and organic concentrations were studied from the nitrogenous balance in the paper. By measuring the nitrogen balance in all experiments under different culture conditions, the removal of total organic carbon and ammonium was positively correlated with total nitrogen removal, especially under high substrate level. With substrate concentration decreasing, ammonium and nitrate removal occurred separately, and ammonium was completely utilized first under low substrate concentration. Compared to that under high substrate level, the specific growth rate of P. mendocina 3-7 was not low under the low substrate level and the pollutant removal efficiencies remained high, which implies the stronger nitrogen removal and acclimatization capacities of the strain in oligotrophic niches.

P5-06-04: Mechanisms of Acquired Resistance to Insulin-Like Growth Factor 1 Receptor Inhibitor in MCF-7 Breast Cancer Cell Line.

Abstract Background: No studies have yet clarified the mechanism of acquired resistance to insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI). Our previous study of 16 breast cancer cell lines found that only MCF-7 expressed high levels of insulin receptor substrate (IRS)-1 and was sensitive to the IGF-1R-TKI NVP-AEW541. Material and Methods: We developed a model of acquired resistance to NVP-AEW541 by continuously exposing MCF-7 cells to NVP-AEW541, naming the model MCF-7-NR. To explore the mechanism of acquired resistance to NVP-AEW541, the effects of NVP-AEW541 on cell growth and IGF-1R signaling in MCF-7 and MCF-7-NR cells were examined. Results: With Western blot analysis, we found that MCF-7-NR had much lower levels of IRS-1 than parental MCF-7. While phosphorylation of Akt was completely inhibited by administration of NVP-AEW541 (3 μM) in both cell lines, phosphorylation of S6K remained high only in MCF-7-NR. The notion of Akt-independent S6K phosphorylation in MCF-7-NR was further supported by the fact that cell growth and phosphorylation of S6K was affected by administration of the Akt inhibitor perifosine to a lesser degree in MCF-7-NR than in MCF-7. Further, the mTOR inhibitor everolimus inhibited phosphorylation of S6K and cell growth equally in both lines. Screening of MCF-7 and MCF-7-NR lines for phosphorylation of 42 receptor tyrosine kinases with and without 3μM NVP-AEW541 showed that Tyro3 phosphorylation remained high only in MCF-7-NR cells. Gene silencing of Tyro3 using siRNA resulted in reduced cell growth, decreased phosphorylation of phosphoinositide-dependent kinase-1 (PDK-1) and protein kinase C α/βII, reduced expression of cyclin D1 in the MCF-7-NR line, with minimal effects evident in the MCF-7 line. Discussion: Findings from our current study support the possibility of predicting sensitivity to NVP-AEW541 by measuring IRS-1 expression, as hypothesized in our previous study. Akt-independent activation of mTOR/S6K by an as-yet-undefined mechanism appears to induce acquired resistance to NVP-AEW541, and as such mTOR inhibitors may have therapeutic value. Tyro3 upregulation and migration of control of cellular growth and cyclin D1 expression may also induce resistance to NVP-AEW541. Although the validity of these findings should be evaluated in further preclinical studies and eventually in clinical trials, our observations may lead to individualized use of NVP-AEW541 and the development of “backup drugs” against tumors that acquire resistance to NVP-AEW541. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-04.

Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels

The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics.

Ubiquitination of Cdc20 by the APC Occurs through an Intramolecular Mechanism

Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the anaphase-promoting complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20's C box. Cdc20 turnover by this intramolecular mechanism is cell cycle regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle.

Discovery and hit-to-lead optimization of novel allosteric glucokinase activators

We report on a hit generation and hit-to-lead program of a novel class of glucokinase activators (GKAs). Hit compounds, activators at low glucose concentration only were identified by vHTS. Scaffold modification reliably afforded activators also at high substrate level. Potency was increased by introduction of a hydrogen bond acceptor as proposed by molecular docking. Replacement of the initial alkylene linkers with a rigid 1,2-phenylene motif followed by further studies eventually furnished a series of potent lead compounds exhibiting steep SAR.