CYP3A4 mediates growth of estrogen receptor-positive breast cancer cells in part by inducing nuclear translocation of phospho-Stat3 through biosynthesis of (±)-14,15-epoxyeicosatrienoic acid (EET)

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G(2)/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ～2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.     

Dephosphorylation of specific sites in the kinase-specificity sequence domain leads to ubiquitin-mediated degradation of the tyrosine phosphatase STEP

STEP (striatal-enriched phosphatase) is a non-receptor tyrosine phosphatase that is specifically expressed in the neurons of the central nervous system. STEP regulates the activity of several effector molecules involved in synaptic plasticity and neuronal cell survival, including MAPKs (mitogen-activated protein kinases), Src family kinases and NMDA (N-methyl-D-aspartic acid) receptors. The critical role of STEP in regulating these effectors requires that its activity be tightly regulated. Previous studies have demonstrated that the activity of STEP is regulated through reversible phosphorylation of a serine residue within the KIM (kinase-interacting motif), by cAMP-dependent PKA (protein kinase A). In the present paper we show that STEP is endogenously phosphorylated at two additional sites located within the KISs (kinase-specificity sequences). The basal activity of ERK (extracellular-signal-regulated kinase) and p38 MAPKs plays an important role in the phosphorylation of these two sites. Dephosphorylation of these two sites leads to polyubiquitination and proteolytic degradation of STEP. Conversely, the proteasome inhibitors MG-132 and epoxomicin can stabilize STEP. The active form of STEP is more susceptible to degradation than the inactive form. Taken together the results of the present paper establish that ubiquitin-dependent proteolysis could be a novel mechanism for irreversibly terminating the activity of STEP.     

Distribution and characterization of ampicillin- and tetracycline-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>from feedlot cattle fed subtherapeutic antimicrobials

Background  

Feedlot cattle in North America are routinely fed subtherapeutic levels of antimicrobials to prevent disease and improve the efficiency of growth. This practice has been shown to promote antimicrobial resistance (AMR) in subpopulations of intestinal microflora including Escherichia coli. To date, studies of AMR in feedlot production settings have rarely employed selective isolation, therefore yielding too few AMR isolates to enable characterization of the emergence and nature of AMR in E. coli as an indicator bacterium. E. coli isolates (n = 531) were recovered from 140 cattle that were housed (10 animals/pen) in 14 pens and received no dietary antimicrobials (control - 5 pens, CON), or were intermittently administered subtherapeutic levels of chlortetracycline (5 pens-T), chlortetracycline + sulfamethazine (4 pens-TS), or virginiamycin (5 pens-V) for two separate periods over a 9-month feeding period. Phenotype and genotype of the isolates were determined by susceptibility testing and pulsed field gel electrophoresis and distribution of characterized isolates among housed cattle reported. It was hypothesized that the feeding of subtherapeutic antibiotics would increase the isolation of distinct genotypes of AMR E. coli from cattle.     

Light intensity determines the extent of mercury toxicity in the cyanobacterium Nostoc muscorum

The extent of mercury (Hg) toxicity in the heterocystous cyanobacterium Nostoc muscorum grown for 72 h in three different light intensities was tested for various physiological parameters viz. growth, pigment contents, photosynthesis, respiration, reactive oxygen species (ROS), malondialdehyde formation and antioxidants. A general reduction in growth and pigments, whole cell O₂-evolution, photosynthetic electron transport activities and ¹⁴CO₂-fixation was observed in a metal concentration–dependent manner, and this effect was more pronounced in high light (130 μmol photon m⁻² s⁻¹)–exposed cells as compared to low (10 μmol photon m⁻² s⁻¹) and normal (70 μmol photon m⁻² s⁻¹) light intensity–exposed cells; however, carotenoids and respiration showed reverse trend. Among photosynthetic electron transport activities, whole chain activity was found to be most sensitive in comparison with photosystem II (PS II) and photosystem I (PS I). Comparing the different photosynthetic processes, ¹⁴CO₂-fixation was most affected in cyanobacterial cells when exposed to Hg and different light intensities. After application of various exogenous electron donors, diphenyl carbazide was found to be more effective to restore PS II activity, suggesting that site of damage lies in between oxygen evolving complex and PS II. Level of oxidative stress (superoxide radical and lipid peroxidation) was maximum at 3.0 μM of Hg when coupled with high light intensity (except hydrogen peroxide). A dose-dependent increase in enzymatic antioxidants such as superoxide dismutase, peroxidase and catalase as well as non-enzymatic antioxidants such as proline, ascorbate, cysteine (except under high light intensity) and non-protein thiols [NP-SH] was observed, which further increased with the increase in light intensity. It was noticed that Hg intoxicates N. muscorum through ROS production, which is aggravated along with the increase in light intensity. Overall results suggest that the severity of the metal stress does increase with Hg concentrations but when coupled with light, it was the light intensity that determines the extent of Hg toxicity.     

High Light Intensity Augments Mercury Toxicity in Cyanobacterium Nostoc muscorum

The present study is aimed at investigating the role of growth irradiance in determining the extent of mercury (Hg) toxicity on various physiological parameters viz. growth, pigment contents, photosynthesis, respiration, ¹⁴CO₂ fixation, photosynthetic electron transport, photorespiration and enzyme activity of cyanobacterium Nostoc muscorum. A general decline was observed in all these parameters with increasing concentration of Hg except for carotenoids content and respiratory activity which exhibited significant enhancement. This effect was more pronounced in high light (130???mol photon m^?2?s^?1) exposed cells as compared to normal (70???mol photon m^?2?s^?1) and low (10???mol photon m^?2?s^?1) light exposed cells. Among the photosynthetic electron transport activities, whole chain was found to be more sensitive than photosystem II (PSII) and photosystem I (PSI). ¹⁴CO₂ fixation was more affected as compared to O₂ evolution when exposed to Hg and different light intensities. Photorespiratory activity, which is an index of protecting organisms from light-induced damage, also showed a similar declining trend. Enzyme assay revealed that among the carboxylating enzymes, activity of RUBISCO was more severely inhibited than PEPCase. Thus, these results suggest that Hg itself was toxic at all tested concentrations and high light intensity augmented its toxicity in N. muscorum inhibiting the growth, pigment contents and photosynthetic activity of the organism.     

Chimeric Vitreoscilla Hemoglobin (VHb) Carrying a Flavoreductase Domain Relieves Nitrosative Stress in Escherichia coli: New Insight into the Functional Role of VHb

Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 μM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.     

Isolation and identification of a new antibacterial entity from the Antarctic cyanobacterium Nostoc CCC 537

The present study was aimed at the isolation, purification and structural elucidation of an antibacterial entity/lead molecule from the Antarctic cyanobacterium Nostoc CCC 537. A methanolic extract of the cyanobacterium was bioassayed with Enterobacter aerogenes as a target. The extract was purified by TLC, and the most active band was subjected to HPLC. The fraction (retention time 15.7 min) designated as the active principle was antibacterial towards Gram positive Mycobacterium tuberculosis H37Rv, Staphylococcus aureus ATCC 25923, Gram negative Salmonella typhi MTCC 3216, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25992, Enterobacter aerogenes MTCC 2822 and multi-drug resistant strains of Escherichia coli GS 2003/01, 02, 03. Based on UV, IR, ¹H NMR, EIMS, and ESIMS data, the structure of the active principle is proposed as 4-[(5-carboxy-2-hydroxy)-benzyl]-1,10-dihydroxy-3,4,7,11,11-pentamethyloctahydrocyclopenta<a>naphthalene (Mr 428, Mp 243–249°C). This intracellular biomolecule is similar to anthraquinone and indane derivatives of a diterpenoid. The rate of production of the active principle currently corresponds to 1.70 mg g⁻¹ biomass dry weight. The inherent property of Nostoc sp. to synthesise niche-specific biomolecules/lead molecules may be exploited for future drug development.     

Homology modeling of phosphoryl thymidine kinase of enterohemorrhagic Escherichia coli OH: 157

Enterohemorrhagic Escherichia coli (EHEC) are source of emerging infectious disease in India. Escherichia coli O157:H7 is an EHEC strain showing multiple antibiotic resistances and the cause of infantile diarrhea and hemolytic uremic syndrome worldwide. A novel strategy to counteract multiple antibiotic resistant organisms is to design drugs which specifically target metabolic pathways such as thiamine biosynthetic pathways found exclusively in prokaryotes. Homology modeling was used for model building of a terminal thiamine biosynthesis enzyme phosphoryl thymidine kinase (Thi E) using Geno3D, Swiss Model and Modeller. The best model was selected based on overall stereochemical quality. The potential ligand binding sites in the model were identified by CASTp server. The validated theoretical model of the 3D structure of the thiE protein of E. coli O157:H7 was predicted using a thiamine phosphate pyrophosphatase from Pyrococcus furiosus (PDB ID: 1X13_A) as template. The active pockets of ligand binding sites in the enzyme were identified. In this study, phosphoryl thymidine kinase (thi E), a terminal enzyme in the thiamine biosynthesis pathway in the pathogen has been modeled to be used in future as a potential drug target by the design of suitable inhibitors.