Morphometrical analysis of the gap-junctional area in parenchymal cells of the rat liver after administration of dibutyryl cAMP and aminophylline

Summary In view of the presumed involvement of gap junctions in the coordination of metabolic activities, the influence of cAMP as a regulatory signal of cell metabolism on gap junctions of hepatocytes has been examined. Male rats received two intraperitoneal doses of 10 mg dibutyryl cAMP/100 g body weight with a time interval of 2.5 h and were decapitated 2.5 h later. After this 5-h interval, analysis of freeze-fracture replicas of fixed liver tissue revealed an increase in the mean (± SEM) gap-junctional membrane portion on the lateral hepatocyte membranes from 0.049 + 0.003 (n = 66) in controls to 0.061 ± 0.003 (n = 70) in treated rats, while the configuration of the connexons appeared unaltered. This effect could not be reinforced by prior administration of aminophylline: the relative gapjunctional area is similarly extended from 0.054 ± 0.003 (n = 126) in the control group to 0.065 ± 0.004 (n = 105) in the experimental animals. Probing for the time course of the junctional response, a group of rats was sacrificed 3 h after the onset of treatment. Already within this time, the gapjunctional area is augmented from 0.042 ± 0.004 (n = 63) in the concurrent controls to 0.069 ± 0.006 (n = 42) in the treated rats. These statistically significant increases in area may suggest a stimulating effect of cAMP on gap junctions of hepatocytes in vivo.This investigation was supported by grant No. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Suberized bundle sheaths in grasses (Poaceae) of different photosynthetic types. II. Apoplastic permeability

Summary The relative hydraulic conductivities of major and minor longitudinal veins, and the apoplastic permeability of the bundle sheaths surrounding all longitudinal and transverse veins were investigated in representatives of the C₃, C₄/NAD-ME, C₄/NAD-ME/PCK intermediate, C₄/PCK and C₄/NADP-ME photosynthetic types. Using the Hagen-Poiseuille equation and measurements of tracheary element diameters, the number of elements in each vein type and the numbers of each vein type, we calculated that 87–99% of the water flow in a longitudinal direction would be expected to occur in the major veins. The permeability of the mestome sheaths and parenchymatous bundle sheaths surrounding the veins was tested using the negatively-charged, fluorescent dye, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS). This dye proved nontoxic to plant tissue at a concentration of 0.5%, according to a deplasmolysis test with onion epidermal strips. The PTS concentration achieved in the tested grass leaves was about 0.035%, well below the toxic limit. When a solution of PTS was fed to the leaves by means of a basal cut, the dye moved into the veins of all orders. From there, it moved outward into the surrounding tissues, indicating that the sheaths surrounding the veins of all orders in all species tested were permeable. Therefore, contrary to previous predictions based on structural observations and some tracer studies, bundle sheaths with suberized cell walls do not function as endodermal layers.     

Leukemic histiocytic sarcoma in a captive common squirrel monkey (Saimiri sciureus) with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus) and Squirrel monkey retrovirus (β-Retrovirus) coinfection

Hematopoietic neoplasia other than lymphoma and leukemia is uncommon among non-human primates. Herein, we provide the first evidence of occurrence of leukemic histiocytic sarcoma in a captive common squirrel monkey with Saimiriine Gammaherpesvirus 2 (Rhadinovirus), Saimiri sciureus lymphocryptovirus 2 (Lymphocryptovirus), and Squirrel monkey retrovirus (β-Retrovirus) coinfection.     

De novo indol-3-ylmethyl glucosinolate biosynthesis,and not long-distance transport,contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.     

Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase

Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.     

Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa

The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright “on” and dark “off” states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0‐Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand β8 containing Thr159 as well as an altered A‐C dimer interface involving strands β7, β8, β10, and β11. The Met159Thr mutation increases the cavity volume for the p‐hydroxybenzylidene‐imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences. Proteins 2015; 83:397–402. © 2014 Wiley Periodicals, Inc.     

A Forward Genetic Screen Targeting the Endothelium Reveals a Regulatory Role for the Lipid Kinase Pi4ka in Myelo- and Erythropoiesis

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Similarity in Several Properties of Psychrophilic Bacteria Grown at Low and Moderate Temperatures

Several properties of psychrophilic pseudomonads were studied with cells grown in batch culture in nutrient broth at 2 and 30 C. No differences were observed in the size, catalase activity, deoxyribonucleic acid, ribonucleic acid, or protein content of cells grown at either temperature. The importance of comparing physiologically similar cells is discussed.     

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection.