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.     

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.     

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H₂O₂ generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations.     

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.     

Heat-shock proteins 70 kDa and 19 kDa are not required for induction of embryogenesis of Brassica napus L. cv. topas microspores

It is currently accepted that 'stress' triggers induction of microspore embryogenesis, and for Brassica napus L. cv. Topas it is heat-shock. It has been postulated that the heat-shock proteins (HSPs) generated during heat stress have a central role in the induction mechanism. To test this hypothesis we developed a microspore induction procedure, using colchicine instead of heat treatment. The level of HSP70 increased significantly during and following the microspore heat treatment while sHSP19 expression was induced at the onset of heat-shock and declined after 8 h. In contrast, induction of embryogenesis with colchicine was not accompanied by elevation of HSP70 nor by induction of sHSP19, indicating that these HSPs are not required for induction of microspore embryogensis in this model system. These data refute the current hypothesis that HSPs have an essential role in triggering microspore embryogenesis.     

Plant cell wall removal: Cause for microtubule instability and division abnormalities in protoplast cultures?

Cell wall removal from plant cells can destabilize the cortical microtubules (MTs) in isolated protoplasts. The degree of destabilization depends on the origin and physiological condition of the cells, enzyme purity and digestion protocol, and the presence, in the digestion medium, of stabilizing factors such as Ca²⁺ or taxol. Disorientation of MTs in protoplasts and the absence of a "normal' cell wall during early culture periods results in abnormalities in mitotic spindles, phragmoplasts, new cross-walls and chromosome segregation. These abnormalities are greatly reduced in older protoplast cultures, where a substantial cell wall had regenerated. It is suggested that the cell wall may serve to stabilize MTs through transmembrane proteins and may play a role in the spatial organization of MT nucleating sites.     

Cytological abnormalities and aberrant microtubule organization during early divisions in mesophyll protoplast cultures of Medicago sativa and Nicotiana tabacum

Karyokinetic and cytokinetic irregularities were examined in cultured mesophyll protoplasts of Medicago sativa L. cv. Regen S and Nicotiana tabacum L. cv. Wisconsin 38 during the early cell divisions. Spindle anomalies, laggine chromalids during anaphase, displaced phragmoplasts during telophase and incomplete cross-walls following the completion of cytokinesis were scored in these cultures. The abnormalities, however, occurred at low frequencies when compared to those found in suspension culture-derived protoplasts of Vicia hajastana . It is suggested that abnormalities are reduced because the mesophyll protoplasts regenerate a substantial cell wall prior to the onset of the first division.
An entirely different class of cytological aberration occurring in 40–50% of the cells was caused by localized protruding of the cytoplasm (budding). The nucleus was often positioned at the site of constriction of the bud. The association of budding with mitosis has led us to postulate a mechanism which may be a cause of budding and to suggest a possible way to prevent it.     

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.