Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Prostatic irradiation-induced sexual dysfunction: A review and multidisciplinary guide to management in the radical radiotherapy era (Part II on Urological Management)

Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related death in men. Radiotherapy is a curative option that is administered via external beam radiation, brachytherapy, or in combination. Sexual dysfunction is a common toxicity following radiotherapy, similar to men undergoing radical prostatectomy, but the etiology is different. The pathophysiology of radiation-induced sexual dysfunction is multi-factorial, and the toxicity is a major cause of impaired quality of life among long-term prostate cancer survivors. Management of a patient’s sexual function during and after radiotherapy requires multidisciplinary coordination of care between radiation oncology, urology, psychiatry, pharmacy, and dermatology. This review provides a framework for clinicians to better understand prostatic radiotherapy-induced sexual dysfunction diagnosis, evaluation, and a patient-centered approach to toxicity preventive strategies and management.     

Agrobacterium bohemicum sp. nov. isolated from poppy seed wastes in central Bohemia

Two non-pathogenic strains R89-1 and R90^T isolated from poppy seed (Papaver somniferum L.) wastes were phenotypically and genotypically characterized. Multilocus sequence analysis (MLSA) was conducted with six genes (atpD, glnA, gyrB, recA, rpoB, 16S rRNA). The strains represented a new species which clustered with Agrobacterium rubi NBRC 13261^T and Agrobacterium skierniewicense Ch11^T type strains. MLSA was further accompanied by whole-genome phylogeny, in silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses for both strains. ANI and dDDH values were deep below the species delineation threshold. Phenotypic features of the novel strains unequivocally allowed their differentiation from all other Agrobacterium species. Unlike other agrobacteria, the strains were salt sensitive and were able to biotransform morphine alkaloids. The dominant cellular fatty acids are 18:1 w7c, 16:0 and 12:0 aldehyde/16:1 iso I/14:0 3OH summed in feature 2 and the major respiratory quinine is Q-10 (87%). The DNA G + C content is 56 mol%. Microbial community analysis indicated probable association with P. somniferum plant material. Altogether, these characteristics showed that strains R90^T and R89-1 represent a new species of the genus Agrobacterium which we propose to name Agrobacterium bohemicum. The type strain of A. bohemicum is R90^T (=CCM 8736^T = DSM 104667^T).     

TLR-7 agonist attenuates airway reactivity and inflammation through Nrf2-mediated antioxidant protection in a murine model of allergic asthma

Toll-like receptors (TLRs) through innate immune system recognize pathogen associated molecular patterns and play an important role in host defense against bacteria, fungi and viruses. TLR-7 is responsible for sensing single stranded nucleic acids of viruses but its activation has been shown to be protective in mouse models of asthma. The NADPH oxidase (NOX) enzymes family mainly produces reactive oxygen species (ROS) in the lung and is involved in regulation of airway inflammation in response to TLRs activation. However, NOX-4 mediated signaling in response to TLR-7 activation in a mouse model of allergic asthma has not been explored previously. Therefore, this study investigated the role TLR-7 activation and downstream oxidant–antioxidant signaling in a murine model of asthma. Mice were sensitized with ovalbumin (OVA) intraperitoneally and treated with TLR-7 agonist, resiquimod (RSQ) intranasally before each OVA challenge from days 14 to 16. Mice were then assessed for airway reactivity, inflammation, and NOX-4 and nuclear factor E2-related factor 2 (Nrf2) related signaling [inducible nitric oxide synthase (iNOS), nitrotyrosine, lipid peroxides and copper/zinc superoxide dismutase (Cu/Zn SOD)]. Treatment with RSQ reduced allergen induced airway reactivity and inflammation. This was paralleled by a decrease in ROS which was due to induction of Nrf2 and Cu/Zn SOD in RSQ treated group. Inhibition of MyD88 reversed RSQ-mediated protective effects on airway reactivity/inflammation due to reduction in Nrf2 signaling. SOD inhibition produced effects similar to MyD88 inhibition. The current study suggests that TLR-7 agonist is beneficial and may be developed into a therapeutic option in allergic asthma.     

Fish models in prion biology: Underwater issues

Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents - prions - are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these and other basic concepts, fundamental questions in prion biology remain unanswered, such as the physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited attention, despite the fact that these animals possess bona fide PrPs. Recent findings, however, have brought fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Although still in its infancy, the use of fish models in TSE research could significantly improve our basic understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Effect of cyanobacterial extracellular products on high-frequency in vitro induction and elongation of Gossypium hirsutum L organs through shoot apex explants

The challenging task of bringing high efficiency transformed plants attracts lot of attention in recent times. In search for this, there have been many attempts made using, different techniques like tissue culture and plant breeding methods. Here we report a suitable alternative facile route, where cyanobacterial extracellular products are utilized as growth regulators and its performance validated on Gossypium hirsutum L. MS medium is tested with cyanobacterial extracellular products of Nostoc ellipsosporum, Dolichospermum flos-aquae and Oscillatoria acuminata .Our best results show that the addition of O. acuminata extracellular product with plant growth hormones gives the excellent induction and elongation in cotton. In addition to this, the multiple shoot was obtained on MS medium fortified with 1.0 mg L^?1 BA with 8% O. acuminata and 1.5 mg L^?1 TDZ with 12% O. acuminata. High frequency of shoot elongation supplemented with MS medium, iP 2.5 mg L^?1 and 16% O. acuminata and root production MS medium fortified with 12% O. acuminata best responsible for regeneration in cotton plants. The rooted plants were hardened and transferred to soil with 90% survival rate.     

Mediation of adenylyl cyclase sensitization by PTX-insensitive GalphaoA, Galphai1, Galphai2 or Galphai3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Galphai/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Galphai/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (Galpha ), were used to determine whether each of the Galphai/o subtypes is able to mediate sensitization of AC. Galpha , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Galphai/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). Each Galphai/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Galphai/o subtype, but the level of sensitization was increased in clones expressing higher levels of Galphai/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Galphai/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC activity.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

适当浓度的过氧化氢诱导人支气管上皮细胞发生钙振荡

包括过氧化氢（Hzoz）在内的活性氧通过引起细胞内钙的变化而造成细胞损伤。然而,不同浓度的H202可以导致细胞内不同的钙变化,并激活不同的信号通路。细胞内钙振荡是其中的一种钙信号变化形式,钙振荡可以调控转录因子NF—KB的活性。该研究探讨可以诱导支气管上皮细胞内钙振苏发生的H2o2浓度。体外培养人支气管上皮细胞,采取钙离子荧光探针Fura_2标记细胞。并使用离子成像系统,观测不同浓度的H：0：（0～1000μmol／L）作用下细胞内钙浓度的变化。结果发现,低于50μmol／L的H202仅仅引起“钙火花”;50～500μmol／L的H202导致细胞内钙振荡的发生;而1000μmol／L的H202引起细胞内持续的高钙;同时也证实150μmol／L的H202诱发明显的钙振荡,而钙振荡随后引起了NF—KB活性的升高。该研究提示,适当浓度的H：0：可以诱发支气管上皮细胞内钙振荡的发生,推测可能是活性氧导致慢性气道炎症损伤的一个机制。