Evaluation of procedures for amplification of small-size samples for hybridization on microarrays

Various approaches have been developed for the preparation of samples for gene expression monitoring. For Affymetrix chips, a standard protocol is widely used; however, this is inefficient for small samples such as laser capture microdissections. Several amplification procedures for such samples already exist, and our goal was to test two of them: the first is based on random PCR amplification, and the second, linear amplification, involves performing the standard protocol twice. We analyzed a dilution of a commercially available mouse brain total RNA preparation and microdissections from mouse hippocampus and striatum. We evaluated the quality of microarray data by analyzing several chip parameters and performing multiple comparisons. At the biological level, brain microdissections prepared with either method gave similar expression results. At the technical level, analysis of the commercial sample showed that random PCR amplification is more reproducible, requires smaller RNA input, and generates cRNA of higher quality than linear amplification.     

β3-adrenergic receptor activation plays an important role in the depressed myocardial contractility via both elevated levels of cellular free Zn2+ and reactive nitrogen species

Role of β₃-AR dysregulation, as either cardio-conserving or cardio-disrupting mediator, remains unknown yet. Therefore, we examined the molecular mechanism of β₃-AR activation in depressed myocardial contractility using a specific agonist CL316243 or using β₃-AR overexpressed cardiomyocytes. Since it has been previously shown a possible correlation between increased cellular free Zn²⁺ ([Zn²⁺]_i) and depressed cardiac contractility, we first demonstrated a relation between β₃-AR activation and increased [Zn²⁺]_i, parallel to the significant depolarization in mitochondrial membrane potential in rat ventricular cardiomyocytes. Furthermore, the increased [Zn²⁺]_i induced a significant increase in messenger RNA (mRNA) level of β₃-AR in cardiomyocytes. Either β₃-AR activation or its overexpression could increase cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels, in line with significant changes in nitric oxide (NO)-pathway, including increases in the ratios of pNOS3/NOS3 and pGSK-3β/GSK-3β, and PKG expression level in cardiomyocytes. Although β₃-AR activation induced depression in both Na⁺- and Ca²⁺-currents, the prolonged action potential (AP) seems to be associated with a marked depression in K⁺-currents. The β₃-AR activation caused a negative inotropic effect on the mechanical activity of the heart, through affecting the cellular Ca²⁺-handling, including its effect on Ca²⁺-leakage from sarcoplasmic reticulum (SR). Our cellular level data with β₃-AR agonism were supported with the data on high [Zn²⁺]_i and β₃-AR protein-level in metabolic syndrome (MetS)-rat heart. Overall, our present data can emphasize the important deleterious effect of β₃-AR activation in cardiac remodeling under pathological condition, at least, through a cross-link between β₃-AR activation, NO-signaling, and [Zn²⁺]_i pathways. Moreover, it is interesting to note that the recovery in ER-stress markers with β₃-AR agonism in hyperglycemic cardiomyocytes is favored. Therefore, how long and to which level the β₃-AR agonism would be friend or become foe remains to be mystery, yet.     

Association of fragile site-associated (FSA) gene expression with epithelial differentiation and tumor development

A novel gene designated as fragile site-associated (FSA) gene was recently identified by positional cloning from the CHO 1q31 fragile site which plays an important role in regulating amplification of multidrug resistance (mdr1) gene in multidrug-resistant cells. FSA produces a message of approximately 16 kb which encodes an open-reading frame of 5005 amino acids. FSA shares sequence similarity with that in Caenorhabditis elegans lpd-3, a lipid storage gene. Using immunohistochemical staining and RNA in situ hybridization we report here that expression of FSA is associated with developmental programs of spermatogenesis and mammary gland in mice. Real-time RT-PCR results also support the upregulation of FSA expression in mammary gland development. Expression of FSA in many tissues including colon, skin, ovary, prostate, and bladder is mainly in the postmitotic, well-differentiated compartments. Moreover, levels of FSA expression are downregulated in tumors of these tissue origins. These results suggest that FSA also plays important roles in regulating mammalian epithelial growth and differentiation and tumor development.     

Preliminary clinical observations with recombinant interleukin-2 in patients with AIDS or LAS

The toxicity of recombinant Interleukin-2 (IL-2) was studied in patients with acquired immunodeficiency syndrome (AIDS) or persistent lymphadenopathy syndrome (LAS). Increasing doses of the drug from 10(3) Units/m2 to 10(6) U/m2 were given as an intravenous bolus injection. At the high-dose levels some minor effects, such as fever up to 39.5 degrees C, chills, malaise or vomiting, were observed. The administration of 10(6) U/m2 as a 4-hour infusion showed identical results. No particular alterations of laboratory parameters were found. At the high-dose level the serum concentration of neopterin, which is released from macrophages after interferon gamma stimulation, was significantly (p less than 0.001) elevated above pretreatment levels. The clinical observation of daily infusions of 10(6)/m2 for 14 days revealed the same side effects. All patients developed lymphocytosis and eosinophilia. Two patients had suffered from severe diarrhoea for several weeks presumably due to cryptosporidiosis. In both cases diarrhoea ceased under the treatment with IL-2 and did not occur in the following two months.     

Effects of experimentally altered glutathione levels on life span, metabolic rate, superoxide dismutase, catalase and inorganic peroxides in the adult housefly, Musca domestica

Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration.     

The effect of synthesis media on the properties of substituted polyaniline/chitosan composites

Substituted polyaniline/chitosan (sPANI/Ch) composites were chemically synthesized in H₂SO₄ and CH₃COOH synthesis media. Structural and physical properties of the composites were characterized by using FTIR, SEM, TGA, UV–vis, XRD techniques, and conductivity measurements. The effect of synthesis media on morphology, thermal stability, conductivity, and crystalline properties was investigated. Chemical interactions between substituted polyanilines and chitosan were explained using FTIR spectra results. The different morphological surfaces were observed in SEM images of the composites. The size of the substituted polyaniline/chitosan (sPANI/Ch) composites was in nanoscale, and the composites synthesized in acetic acid media showed smaller structures than those of H₂SO₄ media and pure chitosan. It was interpreted from XRD results that the composites have amorphous structure and the PNEANI/Ch–CH₃COOH composite has the highest crystallinity.     

Aerosol Exposure to Rift Valley Fever Virus Causes Earlier and More Severe Neuropathology in the Murine Model,which Has Important Implications for Therapeutic Development

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that can cause severe disease including acute-onset hepatitis, delayed-onset encephalitis, retinitis and blindness, or a hemorrhagic syndrome. Currently, no licensed vaccine or therapeutics exist to treat this potentially deadly disease. Detailed studies describing the pathogenesis of RVFV following aerosol exposure have not been completed and candidate therapeutics have not been evaluated following an aerosol exposure. These studies are important because while mosquito transmission is the primary means for human infection, it can also be transmitted by aerosol or through mucosal contact. Therefore, we directly compared the pathogenesis of RVFV following aerosol exposure to a subcutaneous (SC) exposure in the murine model by analyzing survival, clinical observations, blood chemistry, hematology, immunohistochemistry, and virus titration of tissues. Additionally, we evaluated the effectiveness of the nucleoside analog ribavirin administered prophylactically to treat mice exposed by aerosol and SC. The route of exposure did not significantly affect the survival, chemistry or hematology results of the mice. Acute hepatitis occurred despite the route of exposure. However, the development of neuropathology occurred much earlier and was more severe in mice exposed by aerosol compared to SC exposed mice. Mice treated with ribavirin and exposed SC were partially protected, whereas treated mice exposed by aerosol were not protected. Early and aggressive viral invasion of brain tissues following aerosol exposure likely played an important role in ribavirin''s failure to prevent mortality among these animals. Our results highlight the need for more candidate antivirals to treat RVFV infection, especially in the case of a potential aerosol exposure. Additionally, our study provides an account of the key pathogenetic differences in RVF disease following two potential exposure routes and provides important insights into the development and evaluation of potential vaccines and therapeutics to treat RVFV infection.