Microtubule organization during the development of the mitotic apparatus in cultured mesophyll protoplasts of higher plants – an immunofluorescence microscopic study

Microtubule organization in the course of the interphase-mitosis transition, karyokinesis and cytokinesis in cultured mesophyll protoplasts of Medicago sativa L. cv. Regen S and Nicotiana tabacum L. cv. Wisconsin 38 was investigated during the early culture stages. A prominent circular band of microtubules, the prophase band of microtubules (PB) was seen in the cell cortex during prophase in most cells. The band appeared during very early prophase and disappeared during late prophase. Other prophase microtubule arrays were nuclear-envelope associated microtubule fluorescence (perinuclear fluorescence, PNF) and groups of microtubules radiating from the nuclear envelope into the cytoplasm. These radiating microtubutes (perinuclear radiating microtubules, PNR) were particularly long and prominent in the PB plane appearing to link the PNF with the PB network. The possible functions of the PNR are discussed. The role of the PB is discussed in the light of the unorganized nature of these cells.     

Cellular changes during heat shock induction and embryo development of cultured microspores ofBrassica napus cv. Topas

Summary Brassica napus cv. Topas microspores, isolated and cultured near the time of the first pollen mitosis and subjected to a heat treatment of 24 h, can be induced to develop into haploid embryos. This is a study of microspore structure during induction and embryo determination. Early during the 32.5 °C incubation period the nucleus moved away from the edge of the cell, and granules, 30 to 60 nm in diameter, appeared in the mitochondria and as a cluster in the cytoplasm. Cells divided symmetrically and at the end of the heat treatment, acquired the features of induced bicellular structures described previously. The features persisted as the cells divided randomly within the exine for 4–7 days following heat induction. Multicellular structures released from the exine underwent periclinal divisions resulting in protoderm differentiation of the globular embryo, thus determining embryo development. The cytoplasm of early heart-stage embryos contains abundant polyribosomes. Non-embryogenic development was indicated by large accumulations of starch and/or lipid and thickened cell walls or an unorganized pattern of cell division following release of the multicellular structures from the exine. Embryogenesis is discussed in terms of induction, embryo determination and development.     

Induction of embryogenesis with colchicine instead of heat in microspores ofBrassica napus L. cv. Topas

Prior to this report, heat treatment (32.5°C, 24 h) was the method used to induce embryogenesis fromBrassica napus microspores. Continuous culture at 25°C results in pollen development. This study shows that colchicine alone, at the non-inductive temperature of 25°C, can induce embryogenesis, thus demonstrating that heat shock is not required for embryogenic induction inB. napus cv. Topas. Embryogenic frequencies of over 15% were obtained by culturing isolated microspores with 25 M colchicine for 42 h at 25°C. The microspore developmental stages responsive to colchicine were unicellular vacuolate and late unicellular, somewhat earlier stages than the population responsive to heat induction. Other groups have reported that heat-shock proteins are essential to the induction of embryogenesis. The present study offers a method of embryogenic induction without the use of heat which will allow discrimination between the factors associated with response to heat shock and those involved with changing cell development.Abbreviations LU Late-unicellular - PPB Preprophase band - UV unicellular-vacuolate The authors wish to thank C. Bornman for his interest and encouragement. We gratefully acknowledge support from the School of Graduate Studies and Research, Queen's University to J.-P. Z., from Hilleshog AB, Sweden to D.H.S., and from the Natural Sciences and Engineering Research Council of Canada to D.H.S. and W.N. Plant Research Centre contribution No. 1595.     

Effect of novel NOP receptor ligands on food intake in rats

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the NOP opioid receptor, stimulates feeding in rats. The present study evaluated the effect of three newly synthesized NOP receptor agonists and two NOP receptor antagonist on food intake. Freely feeding rats were tested with intracerebroventricular (ICV) injections of the NOP receptor agonists OS-500, OS-462 and OS-461. OS-500 and OS-462 evoked a hyperphagic effect more potent and far more pronounced than that of N/OFQ, while OS-461 was ineffective. OS-500 and OS-462 were also tested by intraperitoneal injection, but were unable to evoke hyperphagia following this route of administration. The NOP receptor antagonist NC-797 and UFP-101 did not modify feeding in freely feeding rats while fully antagonized the hyperphagic effect of N/OFQ. Pre-treatment with UFP-101 but not with NC-797 antagonized the hyperphagic effect of OS-462 and OS-500. The present findings indicate that OS-500, OS-462 may act as potent and long-lasting NOP receptor agonists, whereas UFP-101 and NC-797 show antagonistic properties. The higher efficacy of UFP-101 in blocking the hyperphagic effect of OS-462 and OS-500 may be linked to the better pharmacokinetic profile of this antagonist compared to NC-797. Overall, the results indicate that these compounds may represent valuable pharmacological tools to investigate the role of the brain N/OFQ system.     

Repeatability of gait data using a functional hip joint centre and a mean helical knee axis

Repeatability of traditional kinematic and kinetic models is affected by the ability to accurately locate anatomical landmarks (ALs) to define joint centres and anatomical coordinate systems. Numerical methods that define joint centres and axes of rotation independent of ALs may also improve the repeatability of kinematic and kinetic data. The purpose of this paper was to compare the repeatability of gait data obtained from two models, one based on ALs (AL model), and the other incorporating a functional method to define hip joint centres and a mean helical axis to define knee joint flexion/extension axes (FUN model). A foot calibration rig was also developed to define the foot segment independent of ALs. The FUN model produced slightly more repeatable hip and knee joint kinematic and kinetic data than the AL model, with the advantage of not having to accurately locate ALs. Repeatability of the models was similar comparing within-tester sessions to between-tester sessions. The FUN model may also produce more repeatable data than the AL model in subject populations where location of ALs is difficult. The foot calibration rig employed in both the AL and FUN model provided an easy alternative to define the foot segment and obtain repeatable data, without accurately locating ALs on the foot.     

Effects of the somatomedins and insulin on myoblast differentiation in vitro

The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffe's L6 line. Incubation for 48 hr with either insulin or Temin's multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10^?7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.     

Formyl peptide receptor-1 activation enhances intestinal epithelial cell restitution through phosphatidylinositol 3-kinase-dependent activation of Rac1 and Cdc42

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.     

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T₁ progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.     

Review. Neural mechanisms underlying the vulnerability to develop compulsive drug-seeking habits and addiction

We hypothesize that drug addiction can be viewed as the endpoint of a series of transitions from initial voluntary drug use through the loss of control over this behaviour, such that it becomes habitual and ultimately compulsive. We describe evidence that the switch from controlled to compulsive drug seeking represents a transition at the neural level from prefrontal cortical to striatal control over drug-seeking and drug-taking behaviours as well as a progression from ventral to more dorsal domains of the striatum, mediated by its serially interconnecting dopaminergic circuitry. These neural transitions depend upon the neuroplasticity induced by chronic self-administration of drugs in both cortical and striatal structures, including long-lasting changes that are the consequence of toxic drug effects. We further summarize evidence showing that impulsivity, a spontaneously occurring behavioural tendency in outbred rats that is associated with low dopamine D2/3 receptors in the nucleus accumbens, predicts both the propensity to escalate cocaine intake and the switch to compulsive drug seeking and addiction.     

Proinflammatory macrophages impair skeletal muscle differentiation in obesity through secretion of tumor necrosis factor-α via sustained activation of p38 mitogen-activated protein kinase

Obesity is associated with skeletal muscle loss and impaired myogenesis. Increased infiltration of proinflammatory macrophages in skeletal muscle is noted in obesity and is associated with muscle insulin resistance. However, whether the infiltrated macrophages can contribute to obesity-induced muscle loss is unclear. In this study, we investigate macrophage and muscle differentiation markers in the quadriceps (QC), gastrocnemius, tibia anterior, and soleus muscles from obese mice that were fed a high-fat diet for 16 weeks. Then, we examined the effect and mediator of macrophage-secreted factors on myoblast differentiation in vitro. We found markedly increased levels of proinflammatory macrophage markers (F4/80 and CD11c) in the QC muscle compared with the other three muscle groups. Consistent with the increased levels of proinflammatory macrophage infiltration, the QC muscle also showed a significant reduction in the expression of muscle differentiation makers MYOD1 and myosin heavy chain. In in vitro studies, treatment of C2C12 myoblasts with Raw 264.7 macrophage-conditioned medium (CM) significantly promoted cell proliferation and inhibited myoblast differentiation. Neutralization of tumor necrosis factor α (TNF-α) in Raw 264.7 macrophage CM reversed the reduction of myoblast differentiation. Finally, we found that both macrophage CM and TNF-α induced sustained activation of p38 mitogen-activated protein kinase (MAPK) in C2C12 myoblasts. Together, our findings suggest that the increased infiltration of proinflammatory macrophages could contribute toward obesity-induced muscle loss by secreting inflammatory cytokine TNF-α via the p38 MAPK signaling pathway.