Characterization of Pulmonary CYP4B2, Specific Catalyst of Methyl Oxidation of 3-Methylindole

ABSTRACT The selective toxicity of chemicals to lung tissues is predominately mediated by the selective expression of certain pulmonary cytochrome P450 enzymes. This report describes the purification, cloning, and characterization of a unique enzyme, CYP4B2, from goat lung. The purified P450 enzyme was isolated by multistep ion exchange chromatography to electrophoretic homogeneity with an apparent molecular mass of 55,000 Da. Western blotting studies demonstrated that CYP4B enzymes were selectively expressed in lung tissues of rabbits, rats, and mice. Two cDNAs, CYP4B2 and CYP4B2v, were cloned from goat lung tissue. CYP4B2 was predicted to be 511 amino acids and approximately 82% similar to the four known CYP4B1 proteins. Concurrently, a variant of the known human CYP4B1 cDNA, that contained a S207 insertion, was cloned from human lung tissue. The modified recombinant goat CYP4B2 was expressed in Escherichia coli and the enzyme catalyzed the N-hydroxylation of the prototypical substrate 2AF. CYP4B2 preferentially dehydrogenated, rather than hydroxylated, the pneumotoxicant 3-methylindole (3MI) (V max ϭ 4.61 versus 0.83 nmol/nmol of P450/min, respectively). To investigate the relevance of covalent heme binding of CYP4 enzymes in CYP4B2-mediated metabolism of 3MI, a site-directed mutant (CYP4B2/A315E) was evaluated. The mutation had little effect on the V max of either dehydrogenation or hydroxylation but increased the K m , which decreased the catalytic efficiency (V/K) for 3MI. The A315E mutation shifted the absorbance maximum of the enzyme from 448 to 451 nm, suggesting that the electron density of the heme was altered. These results demonstrate that CYP4B2 is highly specific for methyl group oxidation of 3MI, without formation of ring-oxidized metabolites, and seems to be predominately responsible for the highly organ-specific toxicity of 3MI in goats

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer

Metal biosorption onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris: Multi-metal systems

Binary and ternary systems of Ni2+, Zn2+, and Pb2+ were investigated at initial metal concentrations of 0.5, 1.0 and 2.0 mM as competitive adsorbates using Arthrospira platensis and Chlorella vulgaris as biosorbents. The experimental results were evaluated in terms of equilibrium sorption capacity and metal removal efficiency and fitted to the multi-component Langmuir and Freundlich isotherms. The pseudo second order model of Ho and McKay described well the adsorption kinetics, and the FT-IR spectroscopy confirmed metal binding to both biomasses. Ni2+ and Zn2+ interference on Pb2+ sorption was lower than the contrary, likely due to biosorbent preference to Pb. In general, the higher the total initial metal concentration, the lower the adsorption capacity. The results of this study demonstrated that dry biomass of C. vulgaris behaved as better biosorbent than A. platensis and suggest its use as an effective alternative sorbent for metal removal from wastewater. (C) 2012 Elsevier B.V. All rights reserved.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior [0252/10-7, 0304/10-7

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

Background Tumour Carbonic Anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. Methods We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. Results CAIX expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. Conclusions CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.We are grateful for the support of CRUK [grant number C14303/A17197], the Breast Cancer Research Foundation, the Royal Society, Worldwide Cancer Research and the European Research Council [SURVIVE: 723397]. JP-T and SC received support from the Spanish Ministry of Economy and Competitiveness SAF2014-23622

Coordinated Regulation of Metabolic Transporters and Migration/Invasion by Carbonic Anhydrase IX

Hypoxia is a prominent feature of the tumor microenvironment (TME) and cancer cells must dynamically adapt their metabolism to survive in these conditions. A major consequence of metabolic rewiring by cancer cells in hypoxia is the accumulation of acidic metabolites, leading to the perturbation of intracellular pH (pHi) homeostasis and increased acidosis in the TME. To mitigate the potentially detrimental consequences of an increasingly hypoxic and acidic TME, cancer cells employ a network of enzymes and transporters to regulate pH, particularly the extracellular facing carbonic anhydrase IX (CAIX) and CAXII. In addition to the role that these CAs play in the regulation of pH, recent proteome-wide analyses have revealed the presence of a complex CAIX interactome in cancer cells with roles in metabolite transport, tumor cell migration and invasion. Here, we explore the potential contributions of these interactions to the metabolic landscape of tumor cells in hypoxia and discuss the role of CAIX as a hub for the coordinated regulation of metabolic, migratory and invasive processes by cancer cells. We also discuss recent work targeting CAIX activity using highly selective small molecule inhibitors and briefly discuss ongoing clinical trials involving SLC-0111, a lead candidate small molecule inhibitor of CAIX/CAXII

Impact of Part Time Jobs on Indian Students’ Academic Excellence at Pittsburg State University (PSU)

There are several part time employment opportunities for university students. Some students opt for part-time work and some others do not work at all, it depends on their financial status. Many international students are coming from different parts of the world with different currency values. Most of the students are coming from countries with a decrease in currency value compared to the United States currency’s dollar value. It would be tough for students to manage their basic necessities in the United States. The problem is international Indian students with a decrease in currency value are opting for more part time hours which is directly impacting their academic excellence. The choice to decide whether to work, or not work, how frequently to work and the working environments can have a considerable effect on academic excellence. In order to highlight that problem, the proposed research study assesses the international Indian student’s impact of part time jobs on academic grades. This study will use mixed methods research to answer the following research question: How and to what extent a part-time job for 20 hours per week will impact Indian student’s academic performance in Pittsburg State University? According to International PSU office, there are about 548 international students in spring 2015 out of them 91 students are from India and within that 85 are graduate students, within that approximately 18 students opted for part time jobs within the university. The proposed study will use mixed methods research which includes face to face interviews conducted for part time working Indian graduate students in PSU. The data related to the type of part time job, the hours they are spending on part time work, responsibilities they have at their work, and how many credit hours they opted during that time will be gathered. The academic information data from the database at the international office at Pittsburg State University will also be gathered. Interviews will be conducted during the break times. Proper approvals from the international office and my instructor will be obtained conducting face to face interview and survey on Indian part time working students in PSU. The anonymous data will be collected from the students with no part time jobs and students with 0-10, 10-20 and 20 or above part time working hours per week, with differentiating the type of work, credit hours they opted during that time. The data obtained. Will be analyzed and the related statistical figures will be presented.https://digitalcommons.pittstate.edu/posters_2015/1040/thumbnail.jp

Infantile-onset Pompe disease with neutropenia: Treatment decisions in the face of a unique phenotype.

Infantile-onset Pompe disease manifests with early signs of cardiomyopathy during the first few days to weeks of life. We present the case of a newborn born via emergency cesarean section with atrial flutter and moderate biventricular hypertrophy who was diagnosed with Pompe disease on New York State newborn screen. Diagnosis was confirmed with repeat leukocyte acid alpha-glucosidase (GAA) enzyme activity, GAA gene sequencing, urine Hex4, and evaluation of Cross-Reactive Immunological Material (CRIM) status. The patient was also found to be persistently neutropenic which to our knowledge has not been previously reported in the literature in association with Pompe disease. This report highlights the impact that newborn screening had on time to diagnosis and initiation of treatment with enzyme replacement therapy. We also discuss how our patient's concurrent neutropenia impacted decision making related to immune tolerance induction prior to starting enzyme replacement therapy

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion

Conversion of the cellular prion protein PrPC into its pathogenic isoform PrPSc is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti‐prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis‐folding events