Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

Histamine controls food intake in sheep via H1 receptors

Histamine is a well known amine which controls many physiological functions of the CNS, including fluid balance, appetite, thermoregulation, cardiovascular control, learning and the stress response. All these functions are mediated via three well known membrane receptors (H₁, H₂ and H₃) and, in laboratory animals, feeding behavior is under the control of H₁ type. In order to investigate the central effect of histamine on feeding behavior in sheep and the characterization of the receptor involved, two Latin square design experiments were undertaken using four Iranian Nainee rams implanted with intracerebroventricular cannulae. In the first experiment, 12 h fasted (7:00 p.m.–7:00 a.m.) rams in individual pens were infused with 0 (control), 100, 400 and 800 nM of histamine such that each ram received each dose four times on different days. Ten minutes after injection (7:00 a.m.) water and a food container were put in the pens and the consumption of water and food were recorded at 0.5, 1, 3 and 12 h. Results from this experiment revealed that histamine significantly (P < 0.01) suppressed food intake with no effect on water consumption. In the second experiment the use of three specific histamine antagonists: chloropheniramine, ranitidine and thioperamide, showed that the anorexic effect of histamine was significantly (P < 0.01) blocked by chloropheniramine. It is concluded that feeding behavior in sheep is inhibited by histamine acting via H₁ receptors.     

Multiple cyclin kinase inhibitors promote bile acid-induced apoptosis and autophagy in primary hepatocytes via p53-CD95-dependent signaling

Previously, using primary hepatocytes residing in early G(1) phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21(Cip-1/WAF1/mda6) (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.     

Differentiation of presumptive lens ectoderm of the chick in vitro studied with immunofluorescence techniques

The differentiation capacity of presumptive lens ectoderm was studied in the chick by an in vitro technique using the appearance of central nervous system or lens-specific antigens as indicators of differentiation. Handling the explants resulted in 'autodifferentiation' of both antigens, but co-culture with alcohol-killed primitive node or optic cup material could induce much stronger differentiation. Little specificity exists in the reaction and a hypothesis is presented whereby selection between the two differentiation pathways is thought to be due mainly to maturation within the ectoderm and the inducing tissue plays a minor qualitative role.     

Evaluation of isomers of octopamine for in vitro alpha-adrenergic stimulation of the aortic smooth muscle from spontaneously hypertensive rats

Isomers of octopamine were tested for in vitro alpha-adrenergic stimulation of aortic smooth muscle of spontaneously hypertensive rats (SHR). In order to test the response of alpha 1-adrenoceptors to meta-, para-, and ortho-octopamine, alpha 2-adrenoceptors were blocked with 10(-7) M yohimbine, and to measure the response of alpha 2-adrenoceptors the alpha 1-adrenoceptors were blocked with 10(-7) M prazosin. The contractile response of aortic smooth muscle of SHR to stimulation by phenylephrine, m-, p-, and o-isomers of octopamine in the presence of yohimbine was not appreciably altered. However, administration of prazosin severely attenuated the response of muscles of these compounds indicating that like phenylephrine, the isomers of octopamine stimulate mainly alpha 1-adrenoceptors. The attenuation of contractile response to isomers of octopamine in the presence of prazosin was not as pronounced as in the case of phenylephrine. The comparative potencies of phenylephrine, m-, p-, and o-octopamine in the presence of 10(-7) M prazosin were 1:1.2:2.5:0.75, respectively. Thus, it appears that the isomers of octopamine, especially para- and meta-octopamine, play a much more important role in the physiology of vascular smooth muscle than has been thus far perceived.     

Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation

BAY 43-9006 is a kinase inhibitor that induces apoptosis in a variety of tumor cells. Here we report that treatment with BAY 43-9006 results in marked cytochrome c and AIF release into the cytosol, caspase-9, -8, -7, and -3 activation, and apoptosis in human leukemia cells (U937, Jurkat, and K562). Pronounced apoptosis was also observed in blasts from patients with acute myeloid leukemia. These events were accompanied by ERK1/2 inactivation and caspase-independent down-regulation of Mcl-1. Inducible expression of a constitutively active MEK1 construct did not prevent Mcl-1 down-regulation, suggesting that this event is not related to MEK/ERK pathway inactivation. Furthermore, BAY 43-9006 did not induce major changes in Mcl-1 mRNA levels monitored by real-time PCR or Mcl-1 promoter activity demonstrated by luciferase reporter assays, but it did enhance Mcl-1 down-regulation in actinomycin D-treated cells. Inhibition of protein synthesis by cycloheximide or proteasome function with MG132 and pulse-chase studies with [35S]methionine demonstrated that BAY 43-9006 did not diminish Mcl-1 protein stability, nor did it enhance Mcl-1 ubiquitination, but instead markedly attenuated Mcl-1 translation in association with the rapid and potent dephosphorylation of the eIF4E translation initiation factor. Finally, ectopic expression of Mcl-1 in leukemic cells markedly inhibited BAY 43-9006-mediated cytochrome c cytosolic release, caspase-9, -7, and -3 activation, as well as cell death, indicating that Mcl-1 operates upstream of cytochrome c release and caspase activation. Together, these findings demonstrate that BAY 43-9006 mediates cell death in human leukemia cells, at least in part, through down-regulation of Mcl-1 via inhibition of translation.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

Immobilization of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> PT in resistant matrices to environmental stresses: a strategy for continuous removal of heavy metals under extreme conditions

The purpose of this study was to investigate the potential of immobilized lead- and cadmium-resistant Pseudomonas putida strain PT to remove heavy metals from aqueous medium under extreme conditions. The tolerance and accumulation of cadmium and lead ions by strain PT were investigated by minimal inhibitory concentration (MIC) determination and polymerase chain reaction (PCR) of cadA gene, respectively. The surface chemical functional groups of P. putida PT involved in the metal biosorption were identified by Fourier transform infrared (FTIR). Pseudomonas putida PT was immobilized in three matrices include carboxy-methyl cellulose (CMC), rice bran, and a new composite made of alginate, polyvinyl alcohol (PVA), and CaCO₃ to prepare heavy metal adsorbent. The biosorbents were analyzed by SEM, and their metal removal capability was assayed in two consecutive cycles by atomic absorption spectroscopy. The viability of immobilized bacterial cells was determined by flow cytometry during storage at 4 °C and exposure to the environmental stresses (pH and temperature). The results showed that PT strain was resistant up to 10 mM Pb²⁺ and 8 mM Cd²⁺. FTIR analysis revealed that alcohol, sulfur, phosphate, esters, and amide groups played important roles in metal biosorption process and, also change in metabolic reactions like hydration and polyesters accumulation was observed after metal biosorption. The presence of cadA gene, a heavy metal translocating pump-coding gene, indicated the ability of metals bioaccumulation by the PT strain. Immobilized cells in alginate–PVA–CaCO₃ and rice bran showed the highest metal removal efficiency for Pb²⁺ as 75% and Cd²⁺ as 96.7%, respectively. Metal adsorbents were reusable, and the highest removal efficiency in the second cycle was observed in inoculated alginate–PVA–CaCO₃ (79.5% Pb²⁺ and 45% Cd²⁺). Flow cytometric analysis represented that the immobilized cell viability was retained (<?97%) after 4 weeks storage at 4 °C. Viability under two environmental stresses in all matrices was as follows: <?96% at 25 °C, <?87% at 45 °C, <?85% at pH 4,?<?96% at pH 7, and?<?89% at pH 11. The results signify that these metal adsorbents are efficient technological tools for bioremediation even in harsh environmental conditions.